Ovotoxicants 4-vinylcyclohexene 1,2-monoepoxide and 4-vinylcyclohexene diepoxide disrupt redox status and modify different electrophile sensitive target enzymes and genes in Drosophila melanogaster

Abstract

The compounds 4-vinylcyclohexene 1,2-monoepoxide (VCM) and 4-Vinylcyclohexene diepoxide (VCD) are the two downstream metabolites of 4-vinylcyclohexene (VCH), an ovotoxic agent in mammals. In addition, VCM and VCD may be found as by-products of VCH oxidation in the environment. Recently, we reported the involvement of oxidative stress in the toxicity of VCH in Drosophila melanogaster. However, it was not possible to determine the individual contributions of VCM and VCD in VCH toxicity. Hence, we investigated the toxicity of VCM and VCD (10-1000 µM) in flies after 5 days of exposure via the diet. Our results indicated impairments in climbing behaviour and disruptions in antioxidant balance and redox status evidenced by an increase in DCFH oxidation, decreases in total thiol content and glutathione-S-transferase (GST) activity in the flies exposed to VCM and VCD (p<0.05). These effects were accompanied by disruptions in the transcription of the genes encoding the proteins superoxide dismutase (SOD1), kelch-like erythroid-derived cap-n-collar (CNC) homology (ECH)-associated protein 1 (Keap-1), mitogen activated protein kinase 2 (MAPK-2), catalase, Cyp18a1, JAFRAC 1 (thioredoxin peroxidase 1) and thioredoxin reductase 1 (TrxR-1) (p<0.05). VCM and VCD inhibited acetylcholinesterase (AChE) and delta aminolevulinic acid dehydratase (δ-ALA D) activities in the flies (p<0.05). Indeed, here, we demonstrated that different target enzymes and genes were modified by the electrophiles VCM and VCD in the flies. Thus, D. melanogaster has provided further lessons on the toxicity of VCM and VCD which suggest that the reported toxicity of VCH may be mediated by its transformation to VCM and VCD.

Keywords: 4-Vinylcyclohexene 1,2-monoepoxide; 4-Vinylcyclohexene diepoxide; Changes in mRNA levels; Disruption of antioxidant homoeostasis; Reactive oxygen and nitrogen species.

Graphical abstract

Fig. 1

VCM and VCD reduced survival rates of Drosophila melanogaster. (A) 28 days survival of flies treated with VCM and (B) 28 days survival of flies treated with VCD. Data are presented as mean±SEM of three independent biological replicates carried out in duplicates. Each assay was carried out in three independent experiments. ^*p<0.05 vs control.

Fig. 2

VCM and VCD did not alter survival of D. melanogaster, but impaired climbing behaviour after 5 days of exposure. (A) Five days survival rate and (C) negative geotaxis (climbing rate) of flies treated with VCM; (B) 5 days survival rate and (D) negative geotaxis (climbing rate) of flies treated with VCD. Data are presented as mean±SEM of three independent biological replicates carried out in duplicates. Each assay was carried out in three independent experiments. ^*p<0.05 vs control.

Fig. 3

VCM and VCD enhanced RONS generations after 5 days of exposure in Drosophila melanogaster. (A) RONS levels after treatment of flies with VCM for 5 days; (B) RONS levels after treatment of flies with VCD for 5 days. Data are presented as mean±SEM of three independent biological replicates carried out in duplicates. Each assay was carried out in three independent experiments. ^*p<0.05 vs control.

Fig. 4

Changes in antioxidant enzymes activities and total thiol content of D. melanogaster after 5 days of exposure to VCM and VCD. (A) Catalase activity, (C) Total thiols content and (E) GST activity of flies treated with VCM for 5 days; (B) catalase activity, (D) Total thiols content and (F) GST activity of flies treated with VCD for 5 days. Data are presented as mean±SEM of three independent biological replicates carried out in duplicate. ^*p<0.05 vs control. Each assay was carried out in three independent experiments.

Fig. 5

Quantitative real time RT-PCR (qRT-PCR) analyses of SOD1 and CAT gene expressions in D. melanogaster exposed to VCM and VCD for 5 days. (A) SOD1 and (C) cat mRNA levels of flies treated with VCM for 5 days; (B) SOD1 and (D) CAT mRNA levels of flies treated with VCD for 5 days. Data are presented as mean±SEM of three independent biological replicates carried out in quadruplicates. Each assay was carried out in three independent experiments. ^*p<0.05 vs control.

Fig. 6

Quantitative real time RT-PCR (qRT-PCR) analyses of Keap-1 and MAPK-2 mRNA gene expressions of D. melanogaster after 5 days of exposure to VCM and VCD. (A) Keap-1 and (C) MAPK-2 mRNA levels of flies exposed to VCM for 5 days; (B) Keap-1 and (D) MAPK-2 mRNA levels of flies exposed to VCD for 5 days. Data are presented as mean±SEM of three independent biological replicates carried out in quadruplicates. Each assay was carried out in three independent experiments. ^*p<0.05 vs control.

Fig. 7

Quantitative real time RT-PCR (qRT-PCR) analyses of Cyp18a1, JAFRAC 1 and TrxR-1 mRNA gene expressions of D. melanogaster after 5 days of exposure to VCM and VCD. (A) Cyp18a1, (C) JAFRAC 1 and (E) TrxR-1 mRNA levels of flies treated with VCM for 5 days; (B) Cyp18a1, (D) JAFRAC 1 and (F) TrxR-1 mRNA levels of flies treated with VCD for 5 days. Data are presented as mean±SEM of three independent biological replicates carried out in quadruplicates. Each assay was carried out in three independent experiments. ^*p<0.05 vs control.

Fig. 8

VCM and VCD inhibited AChE and δ-ALA-D activities after 5 days of exposure to Drosophila melanogaster. (A) AChE and (C) δ-ALA-D activities of flies exposed to VCM for 5 days; (B) AChE and (D) δ-ALA-D activities of flies exposed to VCD for 5 days. Data are presented as mean±SEM of three independent biological replicates carried out in duplicates. Each assay was carried out in three independent experiments. ^*p<0.05 vs control.

Scheme 1

Metabolism of 4-vinyvlcyclohexene 1,2-monoepoxide (VCM) and 4-vinyvlcyclohexene diepoxide (VCD) in tissues (e.g. liver). VCD: 4-vinylcyclohexene diepoxide; mEH: microsomal epoxide hydrolase; GST: glutathione-S-transferase.

Scheme 2

Summary of Experimental Design.

Scheme 3

Summary of the mechanisms of 4-vinylcyclohexene 1,2-monoepoxide (VCM)- and 4-vinyvlcyclohexene diepoxide (VCD)-induced toxicity in Drosophila melanogaster.