Reactive oxygen species by fungicide mancozeb in Sertoli- germ cell co- culture

Background: Oxidative stress has been extensively studied as a cause of male infertility and excessive levels of reactive oxygen species (ROS) coupled with a deficiency in antioxidants can lead to it. ROS are derived either from internal sources in the body or from external sources such as exposures to X-rays, ozone, cigarette smoking, air pollutants, and industrial chemicals. Exposure to pesticides affects the reproductive system. They can produce free radicals and change antioxidant capacity or free radicals by inducing oxidative stress. Mancozeb is a typical fungicide that has been shown to produce adverse effects and toxicological manifestations in fertilization, damage to liver, kidney, central nervous system and chromosomes of bone marrow cells in mice.Objective: In the present study, we evaluated the potential of mancozeb oxidative stress induction in Sertoli-germ cell co-culture.Materials and Methods: In this study, ROS generation was measured using DCFH-DA. Testes from mic were dissected, de-capsulated and teased in 10 ml PBS. Cells were isolated from the tubules after collagenase treatment and trypsin treatment at 32oC in a water bath with gentle shaking in the presence of DNase I. Cells were grown in complete DMEM/F12 containing 15% FBS in a humidified incubator of 5% CO2 at 37oC. Cells were loaded with 25 μM DCF for 60 min, then treated with the appropriate concentrations of mancozeb (1, 2.5, 3.5 μM) for 3 hr. To investigate induction of oxidative stress, we also used an antioxidant N-acetylcysteine (NAC). The generation of intracellular ROS was quantitatively monitored in a microplate reader.Results: In the presence of 2.5 and 3.5 μM mancozeb, ROS levels was increased in Sertoli-germ cell co-culture.Conclusion: Our results confirm that mancozeb can mediate cellular toxicity through reactive oxygen species generation which can play a role as a mediator of apoptotic cell.

Keywords:

Fungicide , Mancozeb , ROS , Sertoli-germ cell co-culture