Analysis of the impact of three phthalates on the freshwater gastropod Physella acuta at the transcriptional level

Abstract

Plastic pollution is one of the leading environmental problems. Phthalates are widely used plastic additives released into the environment. Although the effects of phthalates on vertebrates have been extensively studied, there is a knowledge gap regarding their effects on invertebrates. This work analyzes the impact of three phthalates, diethyl phthalate (DEP), benzyl butyl phthalate (BBP), and bis-(2-ethylhexyl) phthalate (DEHP), on the gastropod Physella acuta at the molecular level to establish the putative pathways involved in its response to them. By real-time PCR, we obtained the expression profile of 30 genes in animals exposed for 1 week to 0.1, 10, and 1000 μg/L of each phthalate. The genes cover DNA repair, detoxification, apoptosis, oxidative and stress responses, immunity, energy reserves, and lipid transport. The results show that while DEP and DEHP did not alter the mRNA levels, BBP modulated almost all the analyzed genes. It can be concluded that the impact of BBP is extensive at the molecular level. However, it cannot be dismissed that the increase in transcriptional activity is a general response due to this compound's well-known role as an endocrine disruptor. Additional research is needed to elucidate the differences observed in the impact of these compounds on the gastropod P. acuta.

Figure 1

Structure and conserved domains of the identified Physella acuta proteins. Each protein corresponds to an open reading frame from the sequences used in the study. The proteins are shown with the different motifs that characterize them. The domains are defined according to the CCD functional classification of proteins. Some of the genes were not complete, and the discontinuous line indicates the unknown up- and down-stream regions.

Figure 2

Transcript levels of DNA repairing mechanisms (rad21 and rad50), nervous system (AChE), and oxidative stress (Cat, SOD CuZn, and SOD Mn) genes in Physella acuta adults after in vivo exposure to diethyl phthalate (DEP), benzyl butyl phthalate (BBP), and bis-(2-ethylhexyl) phthalate (DEHP) for 1 week at 19 °C. The mRNA levels were normalized using rpL10, Act, PFKFB2, and GAPDH as reference genes. The comparison was performed with the solvent-exposed (EtOH, acetone) controls. Whisker boxes are shown (n = 9 individuals per box). The median is indicated by the horizontal line within the box, and the 25th and 75th percentiles are indicated by the boundaries of the box. The highest and lowest results are represented by the whiskers. The small triangle inside the box denotes the mean, and the outliers are shown (circles). Significant differences to the respective controls (asterisk) are indicated (p < 0.05).

Figure 3

Transcriptional activity of genes related to apoptosis (Casp3 and AIF3) and phase I detoxification (Cyp2u1, Cyp3a7, Cyp4f22, and Cyp72a15) in adult Physella acuta after in vivo exposure to diethyl phthalate (DEP), benzyl butyl phthalate (BBP), and bis-(2-ethylhexyl) phthalate (DEHP) for 1 week at 19 °C. The mRNA levels were normalized using rpL10, Act, PFKFB2, and GAPDH as reference genes. The comparison was performed with the solvent-exposed (EtOH, acetone) controls. Whisker boxes are shown (n = 9 individuals per box). The median is indicated by the horizontal line within the box, and the 25th and 75th percentiles are indicated by the boundaries of the box. The highest and lowest results are represented by the whiskers. The small triangle inside the box denotes the mean, and the outliers are shown (circles). Significant differences to the respective controls (asterisk) are indicated (p < 0.05).

Figure 4

The mRNA levels of genes related to phase II (GSTk1, GSTm1, GSTo1, and GSTt2) and phase III (MRP1/ABCC1) detoxification and hypoxia (HIF1α) in adult Physella acuta after in vivo exposure to diethyl phthalate (DEP), benzyl butyl phthalate (BBP), and bis-(2-ethylhexyl) phthalate (DEHP) for 1 week at 19 °C. The mRNA levels were normalized using rpL10, Act, PFKFB2, and GAPDH were used. The comparison was performed with the solvent-exposed (EtOH, acetone) controls. Whisker boxes are shown (n = 9 individuals per box). The median is indicated by the horizontal line within the box, and the 25th and 75th percentiles are indicated by the boundaries of the box. The highest and lowest results are represented by the whiskers. The small triangle inside the box denotes the mean, and the outliers are shown (circles). Significant differences to the respective controls (asterisk) are indicated (p < 0.05).

Figure 5

The mRNA levels of genes involved in epigenetic regulation (DNMT1, KAT6B, and HDAC1) and stress response (sHsp16.6, sHsp17.9, and Hsp60) in adult Physella acuta after in vivo exposure to diethyl phthalate (DEP), benzyl butyl phthalate (BBP), and bis-(2-ethylhexyl) phthalate (DEHP) for 1 week at 19 °C. The mRNA levels were normalized using rpL10, Act, PFKFB2, and GAPDH as reference genes. Treated animals were compared to solvent-exposed (EtOH, acetone) controls. Whisker boxes are shown (n = 9 individuals per box). The horizontal line within the box indicates the median, and the 25th and 75th percentiles are indicated by the boundaries of the box. The whiskers represent the highest and lowest results. The small triangle inside the box denotes the mean, and the outliers are shown (circles). Significant differences to the respective controls (asterisk) are indicated (p < 0.05).

Figure 6

Modulation of genes related to stress (Hsc70-4, Grp78/BiP, and Hsp90), immune system (ApA), energy metabolism (PYGL), and lipid transportation (ORP8) in adult Physella acuta after in vivo exposure to to diethyl phthalate (DEP), benzyl butyl phthalate (BBP), and bis-(2-ethylhexyl) phthalate (DEHP) for 1 week at 19 °C. The mRNA levels were normalized using rpL10, Act, PFKFB2, and GAPDH as reference genes. Treatments were compared to respective solvent-exposed (EtOH, acetone) controls. Whisker boxes are shown (n = 9 individuals per box). The median is indicated by the horizontal line within the box, and the 25th and 75th percentiles are indicated by the boundaries of the box. The highest and lowest results are represented by the whiskers. The small triangle inside the box denotes the mean and the outliers are shown (circles). Significant differences to the respective controls (asterisk) are indicated (p < 0.05).