Simultaneous exposure to vinylcyclohexene and methylmercury in Drosophila melanogaster: biochemical and molecular analyses

Abstract

Background: Exposure to vinylcyclohexene (VCH) and methylmercury (MeHg⁺) can induce oxidative stress and gene modulation. Several studies have been evaluating the effects of VCH and MeHg⁺, but little is known about interactive effects between them. This work aimed to assess the exposure and co-exposure effects of MeHg⁺ and VCH on oxidative stress and gene modulation in Drosophila melanogaster.

Methods: Reactive species production, glutathione S-transferase (GST) and acetylcholinesterase (AChE) activities were evaluated after exposure and co-exposure to VCH (1 mM) and MeHg⁺ (0.2 mM) for one or three days in the head and body (thorax and abdomen) of flies. The expression of genes related to redox state and inflammatory response was evaluated after exposure and co-exposure to VCH and MeHg⁺ for three days.

Results: Survival decreased only in flies co-exposed to VCH and MeHg⁺ for three days. All treatments increased total reactive species production after one day of exposure. However, no significant changes were observed in the head after three days of exposure. One day of exposure to VCH caused an increase in the head GST activity, whereas MeHg⁺ induced an increase after three days of exposure. Regarding the body, all treatments increased GST activity after one day of exposure, but only the flies exposed to MeHg⁺ presented an increase in GST activity after three days of exposure. Treatments did not alter AChE activity in the head. As for gene expression, there was a significant increase in the Relish transcription factor gene in the flies' body, but Nrf2, Keap1, Jafrac1, TrxR1, and NF-κβ were not altered.

Conclusion: The results suggest that exposure to VCH and MeHg⁺ induce oxidative stress and activation of an inflammatory response in fruit flies.

Keywords: Alternative model; Drosophila melanogaster; Inflammatory response; Oxidative stress; Xenobiotic.

Fig. 1

Kaplan-Meier percent survival of flies after exposure to VCH and MeHg⁺. Percent survival of flies after exposure to different concentrations of MeHg⁺ (0 - 0.4 mM) for four days (a). Percent survival of flies after exposure and co-exposure to VCH and MeHg⁺ for one (b) or three-days (c). Results were analyzed by Logrank test for trend and were considered significantly different when p < 0.05.

Fig. 2

Percent of climbing of flies after exposure and co-exposure to VCH and MeHg⁺ for one (a) or three days (b). Data were expressed as the mean ± standard error. Results were analyzed by Two-way ANOVA (VCH x MeHg⁺ as independent factors) (p > 0.05).

Fig. 3

DCFDA oxidation of flies head after exposure and co-exposure to VCH and MeHg⁺ for one (a and b) or three days (c and d). Kinetic readings were expressed as the mean in a and c (standard errors were omitted for better viewing) and delta per minute as mean ± standard error in b and d). Results were analyzed by four-way ANOVA (VCH x MeHg⁺ x age x time of kinetic reading which was treated as repeated measures) and were considered significantly different when p < 0.05

Fig. 4

DCFDA oxidation of flies body after exposure and co-exposure to VCH and MeHg⁺ for one (a and b) or three days (c and d). Kinetic readings were expressed as the mean in a and c (standard errors were omitted for better viewing) and delta per minute as mean ± standard error in b and d. Results were analyzed by four-way ANOVA (VCH x MeHg⁺ x age x time of kinetic reading which was treated as repeated measures) and were considered significantly different when p < 0.05

Fig. 5

GST activity of flies head after exposure and co-exposure to VCH and MeHg⁺ for one (a and b)or three days (c and d). Kinetic readings were expressed as the mean in a and c (standard errors were omitted for better viewing) and delta per minute as mean ± standard error in b and d. Results were analyzed by four-way ANOVA (VCH x MeHg⁺ x age x time of kinetic reading which was treated as repeated measures) and were considered significantly different when p < 0.05

Fig. 6

GST activity of flies body after exposure and co-exposure to VCH and MeHg⁺ for one (a and b)or three days (c and d). Kinetic readings were expressed as the mean in a and c (standard errors were omitted for better viewing) and delta per minute as mean ± standard error in b and d. Results were analyzed by four-way ANOVA (VCH x MeHg⁺ x age x time of kinetic reading which was treated as repeated measures) and were considered significantly different when p < 0.05

Fig. 7

AChE activity of flies head after exposure and co-exposure to VCH and MeHg⁺ for one (a and b)or three days (c and d). Kinetic readings were expressed as the mean in a and c (standard errors were omitted for better viewing) and delta per minute as mean ± standard error in b and d. Results were analyzed by four-way ANOVA (VCH x MeHg⁺ x age x time of kinetic reading which was treated as repeated measures) and were considered significantly different when p < 0.05

Fig. 8

Expression of the genes encoding Nrf2 and Keap1. mRNA levels of Nrf2 in the head (a) and body (b), as well as, Keap 1 in the head (c) and body (d) of D. melanogaster after exposure and coexposure to VCH and MeHg⁺ for three days. Data are expressed as the mean ± standard error. Results were analyzed by paired t-test and were considered significantly different when p < 0.05.

Fig. 9

Expression of the genes encoding Jafrac1 and TrxR1. mRNA levels of Jafrac1 in the head (a) and body (b), TrxR1 in the head (c) and body (d) of D. melanogaster after exposure and co-exposure to VCH and MeHg⁺ for three days. Data are expressed as the mean ± standard error. Results were analyzed by paired t-test and were considered significantly different when p < 0.05

Fig. 10

Expression of the genes encoding NF-κB activating protein and Relish. mRNA levels of NF-κB activating protein in the head (a) and body (b), Relish in the head (c) and body (d) of D. melanogaster after exposure and co-exposure to VCHand MeHg⁺ for three days. Data are expressed as the mean ± standard error. Results were analyzed by paired t-test and were considered significantly different when p < 0.05