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. 2015 May;4(3):645-654.
doi: 10.1039/C4TX00141A.

Reproductive toxicity and meiotic dysfunction following exposure to the pesticides Maneb, Diazinon and Fenarimol

Daniela A Parodi  1 , Jasmine Sjarif  2 , Yichang Chen  3 , Patrick Allard  4
Affiliations

Reproductive toxicity and meiotic dysfunction following exposure to the pesticides Maneb, Diazinon and Fenarimol

Daniela A Parodi et al. Toxicol Res (Camb). 2015 May.

Abstract

The comprehensive identification and mechanistic analysis of reproductive toxicants constitutes one of the major hurdles in the toxicological assessment of chemicals originating from the large number of chemicals to be tested and the difficulty in examining germ cells at various stages of their development. We previously described the development of an assay in the roundworm Caenorhabditis elegans that allows the detection of chemicals bearing aneugenic activity and that could be used for the detection of germline toxicity. We present here new evidence for the reproductive toxicity of three pesticides identified in our germline toxicity assay: Maneb, Diazinon and Fenarimol. We show that all three pesticides cause an acute germline nuclear loss in exposed nematodes in a dose-dependent fashion. The loss of germline nuclei coincides with the meiotic stage of pachytene during Prophase I and is dependent on the germline apoptotic machinery suggesting activation of a meiotic checkpoint. Further investigation revealed a profound dysregulation of the meiotic program revealed by (1) an alteration of the kinetics of double strand repair, (2) the disruption of the process of chromosome morphogenesis at the end of Prophase I and (3) the reorganization of the meiotic differentiation gradient inherent to the C. elegans germline following exposure to Maneb and Diazinon. These defects correlate with a significant increase in embryonic lethality and a corresponding decrease in the number of progeny. These results therefore provide strong evidence for the reproductive toxicity of Maneb, Diazinon and Fenarimol rooted in the alteration of early steps of germ cell differentiation.

Keywords: Caenorhabditis elegans; aneuploidy; chemical screen; reproductive toxicicity.

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Figures

Fig. 1
Fig. 1. Exposure to Maneb, Diazinon and Fenarimol results in germline cell loss. (A, B) Percentage of worms with germ cell loss in worms exposed to no chemical, vehicle (0.1% DMSO) or pesticides at 100 μM for 24 hours. Error bars (SEM) represent the comparison between DMSO and the pesticide exposed groups. n = 182–132, *p ≤ 0.05 and **p ≤ 0.005. (B) Dose–response of the three positive pesticides showing the percentage of worms with germ cell loss in worms exposed either with vehicle (0.1% DMSO) or pesticides at the following doses in order: 100 μM, 10 μM, 1 μM, 0.1 μM. n = 87–43, *p ≤ 0.05 and **p ≤ 0.005. (C–D) Images of DAPI-stained whole-mount gonads from age-matched hermaphrodites exposed to either (C) DMSO or (D) Maneb at 100 μM. Scale bar, 20 μm. Red arrow indicates the location of the nuclear gap.
Fig. 2
Fig. 2. Exposure to Maneb, Diazinon and Fenarimol results in an increase in apoptosis in the germline. (A) Quantitation of germ cell apoptosis in wild type N2 and ced-3(n717) worms exposed either with vehicle (0.1% DMSO) or pesticides (100 μM) for 24 hours. Loss function of ced-3 blocks germ cell apoptosis induced by the exposure to pesticides. Error bars represent SEM. n = 105–84, **p ≤ 0.005. (B–D) Representative pictures of germ cell apoptosis. Acridine Orange staining of gonads from age-matched hermaphrodites exposed to either (B) DMSO or (C–D) a representative pesticide (Diazinon) in (C) wild type N2 or (D) ced-3(n717) worms. Bright AO staining (red arrows) was observed in the apoptotic germ cells of the wild-type N2 worms. The scale bar represents 20 μm.
Fig. 3
Fig. 3. Exposure to Maneb, Diazinon and Fenarimol results in an activation of the DNA damage checkpoint. Representative immunostaining images for phosphorylated CHK-1(pCHK-1, red) in mid to late pachytene nuclei of dissected gonads exposed to either DMSO (0.1%) or Maned, Diazinon or Fenarimol exposed at 100 μM. n = 8–12. Scale bar, 5 μm.
Fig. 4
Fig. 4. Exposure to Maneb, Diazinon and Fenarimol results in an alteration of the kinetics of meiotic recombination. (A) Mid-late pachytene nuclei from germlines exposed to either DMSO or a representative pesticide (Fenarimol). The pesticide-exposed germlines show elevated levels of RAD-51 (red) foci compared with control. Scale bar = 5 μm. (B) Sketch map showing how gonads were divided into 5 zones along the germline axis for analysis purposes. (C) Histograms depict the quantitation of RAD-51 foci performed in whole-mount gonads from age-matched hermaphrodites exposed to either DMSO, Meneb, Diazinon or Fenarimol. The number of RAD-51 foci per nucleus is categorized by the color code shown on the right. The percentage of nuclei observed for each category (y axis) is depicted for each zone (x axis). Zones 1, 2, 3 , 4 and 5 correspond to mitotic, transition, early, mid- and late pachytene stages respectively.
Fig. 5
Fig. 5. Exposure to Maneb, Diazinon and Fenarimol causes severe defects at diakinesis. (A) Six intact bivalents are observed in control (DMSO, 0.1%) -1 oocytes. By contrast, chromosomes with an frail, fragmented and uncondensed appearance are observed in –1 oocytes in Maneb (100 μM), Diazinon (100 μM) and Fenarimol (100 μM) exposed germlines. Scale bar, 5 μm. (B) Quantification of the chromosome morphology defects observed in bivalents at diakinesis (–1, –2 and –3 oocytes). Error bars represent SEM. n = 57–68, *p ≤ 0.05 and **p ≤ 0.005. (C) Immunocololalizaton of SYP-2 (red) on DAPI-stained (blue) –2 oocytes reveals a delay in SC disassembly and bialent morphogenesis in Maneb, Diazinon and Fenarimol exposed germlines. Scale bar, 5 μm. (D) Percentage of nuclei with expression of SYP-2 in –1 to –5 oocytes. Error bars represent SEM. n = 30, **p ≤ 0.005.
Fig. 6
Fig. 6. Exposure to Maneb, Diazinon and Fenarimol results fertility. (A) Brood size (total progeny number) from hermaphrodites exposed either to vehicle (0.1% DMSO) or to Maneb, Diazinon or Fenarimol (100 μM). Error bars represent SEM. n = 8; *p ≤ 0.05. (B) Fertilized egg number from hermaphrodites exposed either to vehicle (0.1% DMSO) or to Maneb, Diazinon or Fenarimol (100 μM). Error bars represent SEM. n = 8; *p ≤ 0.05. (C) Embryonic lethality of the progeny of hermaphrodites exposed either to vehicle (0.1% DMSO) or to Maneb, Diazinon or Fenarimol (100 μM). Error bars represent SEM. n = 8; *p ≤ 0.05 and **p ≤ 0.005.
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