Toxicological Study and Genetic Basis of BTEX Susceptibility in Drosophila melanogaster

Abstract

Benzene, toluene, ethylbenzene and xylene, also known as BTEX, are released into environmental media by petroleum product exploratory and exploitative activities and are harmful to humans and animals. Testing the effects of these chemicals on a significantly large scale requires an inexpensive, rapidly developing model organism such as Drosophila melanogaster. In this study, the toxicological profile of benzene, toluene, ethylbenzene, p-xylene, m-xylene, and o-xylene in D. melanogaster was evaluated. Adult animals were monitored for acute toxicity effects. Similarly, first instar larvae reared separately on the same compounds were monitored for the ability to develop into adult flies (eclosion). Further, the impact of fixed concentrations of benzene and xylene on apoptosis and mitosis were investigated in adult progenitor tissues found in third instar larvae. Toluene is the most toxic to adult flies with an LC₅₀ of 0.166 mM, while a significant and dose-dependent decrease in fly eclosion was observed with benzene, p-xylene, and o-xylene. An increase in apoptosis and mitosis was also observed in animals exposed to benzene and p-xylene. Through Genome Wide Association Screening (GWAS), 38 regions of the D. melanogaster genome were identified as critical for responses to p-xylene. This study reveals the strength of D. Melanogaster genetics as an accessible approach to study BTEX compounds.

Keywords: BTEX; DGRP; Drosophila melanogaster; GWAS; apoptosis; development; imaginal disk.

FIGURE 1

Quantitation of Dcp-1 and PH3 staining in wing imaginal disks following p-xylene and benzene feeding. (A) Summary of data showing activity of cleaved Dcp-1 in wing disks of w¹¹¹⁸ 3rd instar larva. Data are represented by mean antibody-positive cell count ± SE (N = 8). (B): Summary of data showing activity of PH3 in wing disks of w¹¹¹⁸ 3rd instar larva. Data are represented by mean cell count ± SE (N = 6). Statistical significance, treated versus control group: * (p < 0.05).

FIGURE 2

Visualization of Dcp-1 and PH3 staining in wing imaginal disks following p-xylene and benzene feeding. Wing disks of 3rd instar larvae immunostained for cleaved Dcp-1 (red), PH3 (green), and DAPI to visualize DNA (white). (A–D). Representative images of cell division and apoptosis in wing disk of control flies. First instar larvae were treated with 4 μL of deionized water for 72 h as described in “Materials and Methods” section. (E–H) Representative images of cell death and apoptosis in DMSO treatment. First instar larvae were treated with 0.1% of DMSO for 72 h as described in “Materials and Methods” section. (I–L) Representative images of cell division and apoptosis in p-xylene. First instar larvae were treated with 1.068 mM of p-xylene for 72 h as described in “Materials and Methods” section. (M–P) First instar larvae were treated with 1.068 mM of benzene for 72 h as described in “Materials and Methods” section. Dashed lines indicate shape of Drosophila melanogaster wing disk. Scale bar = 100 μm.

FIGURE 3

Variation in p-xylene eclosion rates among 144 DGRP lines.

FIGURE 4

Quantile-Quantile plot of association analyses of p-xylene resistance and susceptibility among 144 DGRP lines. The red line indicates the expected and the black line the observed p values. Six top performing lines are highlighted.

FIGURE 5

Heatmap of linkage disequilibrium (LD) values (R²) between candidate SNPs. The heat map depicts the degree of LD, r², between variants. The five major chromosome arms are defined by the black lines. Red corresponds to complete LD and blue to absence of LD. A nominal P ≤ 10^{– 5} is indicated with a red line for each trait.