ATM/Chk2 and ATR/Chk1 Pathways Respond to DNA Damage Induced by Movento® 240SC and Envidor® 240SC Keto-Enol Insecticides in the Germarium of Drosophila melanogaster

Abstract

DNA damage response (DDR) pathways in keto-enol genotoxicity have not been characterized, and few studies have reported genotoxic effects in non-target organisms. The present study shows that concentrations of 11.2, 22.4, 37.3 mg/L of Movento^® 240SC and 12.3, 24.6, 41.1 mg/L of Envidor^® 240SC for 72 h oral exposure induced DSBs by significantly increasing the percentage of γH2AV expression in regions 2b and 3 from the germarium of wild type females of Drosophila melanogaster Oregon R, compared to the control group (0.0 mg/L of insecticides), via confocal immunofluorescence microscopy. The comparison between both insecticides' reveals that only the Envidor^® 240SC induces concentration-dependent DNA damage, as well as structural changes in the germarium. We determined that the DDR induced by Movento^® 240SC depends on the activation of the ATM^tefu, Chk1^grp and Chk2^lok kinases by significantly increasing the percentage of expression of γH2AV in regions 2b and 3 of the germarium, and that ATR^mei-29D and p53^dp53 kinases only respond at the highest concentration of 37.3 mg/L of Movento^® 240SC. With the Envidor^® 240SC insecticide, we determined that the DDR depends on the activation of the ATR^mei-29D/Chk1^grp and ATM^tefu/Chk2^lok kinases, and p53^dp53 by significantly increasing the percentage of expression of γH2AV in the germarium.

Keywords: DNA damage response; Drosophila germarium; keto-enol insecticides.

Figure 1

Treatment scheme for keto-enol insecticides Movento^® 240SC and Envidor^® 240SC in Oregon R wild type females and DDR mutants.

Figure 2

Germarium of wild-type Oregon R females immunostained against anti-γH2AV. (A) Schematic image of the germarium. Region 1: TFC, CC, GSCs, GC, EC, Cystoblast. Region 2a: Cysts, FSC. Region 2b: FC and Cyst oocyte. Region 3: FC, first ovarian chamber (first stage of oogenesis). (B1–C8) anti-γH2AV monoclonal antibody (green) to detect DSBs in DNA by immunofluorescence and DAPI for nuclei staining, red immunolocalization of γH2AV in the germarium, scale bar represents 10 μm. (B1,B2,C1,C2) Control group (0.0 mg/L), γH2AV (red) in region 2a of the germarium. (B3–B8) Germarium of females exposed to 11.2, 22.4, 37.3 mg/L of Movento^® 240SC, γH2AV (red) in regions 2b and 3. (C3–C8) Germarium of females exposed to 12.3, 24.6, 41.1 mg/L of Envidor^® 240SC. (C3,C4) γH2AV (red) in region 2a. (C5,C8) γH2AV (red) in regions 2b and 3. (D) Averages of three independent experiments of (%) expression of γH2AV in the germarium of the wild-type strain Oregon R with and without exposure to the keto-enol insecticides Movento^® 240SC and Envidor^® 240SC. *** Significant differences (p < 0.0001), two-way ANOVA and Tukey post hoc test. (E) Linear regression analysis.

Figure 3

(A–E) Germarium of DDR mutant females immunostained against anti-γH2AV after 72 h of exposure to concentrations of 0.0, 11.2, 22.4, and 37.3 mg/L of Movento^® 240SC. Composite image in blue DAPI marking cell nuclei and in green γH2AV, red immunolocalization of γH2AV in the germarium, scale bar represents 10 μm. (A¹) (a.i. Movento^® 240SC). (A) ATM^tefu. (A1,A2) expression of γH2AV in regions 1, 2a, 2b of the germarium. (A3–A8) expression of γH2AV in all regions of the germarium and morphological alterations. (B) ATR^mei−29D. (B1–B4) expression of γH2AV in regions 2a, 2b and 3 of the germarium. (B5–B8) expression of γH2AV in all regions of the germarium. (C) Chk1^grp/Chk2^lok. (C1,C2) expression of γH2AV in regions 2a and 2b of the germarium. (C3,C4) expression of γH2AV in regions 1, 2a and 3 of the germarium and absence of nuclei in regions 1 and 2a (yellow-dotted line). (C5,C6) expression of γH2AV in regions 2a and 2b of the germarium, absence of nuclei in regions 1 and 2a (yellow-dotted line) and morphological changes. (C7,C8) expression of γH2AV in all regions of the germarium, absence of nuclei in regions 1 and 2a (yellow-dotted line) and morphological changes. (D) Chk1^grp. (D1,D2) expression of γH2AV in regions 1, 2a and 2b of the germarium. (D3,D4) expression of γH2AV in all regions of the germarium. (D5,D6) expression of γH2AV in regions 2a and 3 of the germarium. (D7,D8) expression of γH2AV in regions 2a, 2b and 3 of the germarium, absence of nuclei in region 1 (yellow-dotted line) and reduction in the size of the germarium. (E) p53^dp53. (E1–E4) expression of γH2AV in regions 2a and 3 of the germarium. (E5–E8) expression of γH2AV in all regions of the germarium.

Figure 4

(A) Averages of three independent experiments of (%) expression of γH2AV in the germarium of the wild-type strain Oregon R and DDR mutants, with and without exposure to the keto-enol insecticide Movento^® 240SC. *** Significant differences (p < 0.0001), * significant differences (p < 0.005), two-way ANOVA and Tukey post hoc test. (B–F) Linear regression analysis.

Figure 5

(A–E) Germarium of DDR mutant females immunostained against anti-γH2AV after 72 h of exposure to concentrations of 0.0, 12.3, 24.6, and 41.1 mg/L to Envidor^® 240SC. Composite image in blue DAPI marking cell nuclei and in green γH2AV, red immunolocalization of γH2AV in the germarium, scale bar represents 10 μm. (A¹) Chemical structure of Spirodiclofen (a.i. Envidor^® 240SC). (A) ATM^tefu. (A1,A2) expression of γH2AV in regions 1, 2a, 2b of the germarium. (A3,A4) expression of γH2AV in all regions of the germarium. (A5,A6) expression of γH2AV in regions 1, 2a and 2b of the germarium and absence of nuclei in region 3 (yellow-dotted line). (A7,A8) expression of γH2AV in all regions of the germarium. (B) ATR^mei−29D. (B1–B4) expression of γH2AV in all regions of the germarium. (B5,B6) expression of γH2AV in regions 1, 2a and 2b of the germarium, absence of nuclei in region 3 (yellow-dotted line) and morphological changes. (B7,B8) expression of γH2AV in all regions of the germarium. (C) Chk1^grp/Chk2^lok. (C1,C2) expression of γH2AV in regions 2a and 2b of the germarium. (C3,C4) expression of γH2AV in regions 1 and 2a of the germarium and absence of nuclei in regions 1, 2a and 3 (yellow-dotted line). (C5,C6) expression of γH2AV in region 2a of the germarium, absence of nuclei in regions 1, 2a and 3 (yellow-dotted line) and morphological changes. (C7,C8) expression of γH2AV in all regions of the germarium and absence of nuclei in regions 1, 2b and 3 (yellow-dotted line). (D) Chk1^grp. (D1,D2) expression of γH2AV in regions 1, 2a and 2b of the germarium. (D3–D8) expression of γH2AV in all regions of the germarium. (E) p53^dp53. (E1,E2) expression of γH2AV in regions 2a and 3 of the germarium. (E3–E6) expression of γH2AV in all regions of the germarium. (E7,E8) expression of γH2AV in all regions of the germarium, morphological changes and reduction in the size of the germarium.

Figure 6

(A) Averages of three independent experiments of (%) expression of γH2AV in the germarium of the wild-type strain Oregon R and DDR mutants, with and without exposure to the keto-enol insecticide Envidor^® 240SC. *** Significant differences (p < 0.0001), two-way ANOVA and Tukey post hoc test. (B–F) Linear regression analysis.

Figure 7

DNA damage response (DDR) mechanism induced by keto-enol insecticides. (A) DDR induced by Movento^® 240SC. (B) DDR induced by Envidor^® 240SC.