Life-Cycle-Dependent Toxicities of Mono- and Bifunctional Alkylating Agents in the 3R-Compliant Model Organism C. elegans

Abstract

Caenorhabditis elegans (C. elegans) is gaining recognition and importance as an organismic model for toxicity testing in line with the 3Rs principle (replace, reduce, refine). In this study, we explored the use of C. elegans to examine the toxicities of alkylating sulphur mustard analogues, specifically the monofunctional agent 2-chloroethyl-ethyl sulphide (CEES) and the bifunctional, crosslinking agent mechlorethamine (HN2). We exposed wild-type worms at different life cycle stages (from larvae L1 to adulthood day 10) to CEES or HN2 and scored their viability 24 h later. The susceptibility of C. elegans to CEES and HN2 paralleled that of human cells, with HN2 exhibiting higher toxicity than CEES, reflected in LC₅₀ values in the high µM to low mM range. Importantly, the effects were dependent on the worms' developmental stage as well as organismic age: the highest susceptibility was observed in L1, whereas the lowest was observed in L4 worms. In adult worms, susceptibility to alkylating agents increased with advanced age, especially to HN2. To examine reproductive effects, L4 worms were exposed to CEES and HN2, and both the offspring and the percentage of unhatched eggs were assessed. Moreover, germline apoptosis was assessed by using ced-1p::GFP (MD701) worms. In contrast to concentrations that elicited low toxicities to L4 worms, CEES and HN2 were highly toxic to germline cells, manifesting as increased germline apoptosis as well as reduced offspring number and percentage of eggs hatched. Again, HN2 exhibited stronger effects than CEES. Compound specificity was also evident in toxicities to dopaminergic neurons-HN2 exposure affected expression of dopamine transporter DAT-1 (strain BY200) at lower concentrations than CEES, suggesting a higher neurotoxic effect. Mechanistically, nicotinamide adenine dinucleotide (NAD⁺) has been linked to mustard agent toxicities. Therefore, the NAD⁺-dependent system was investigated in the response to CEES and HN2 treatment. Overall NAD⁺ levels in worm extracts were revealed to be largely resistant to mustard exposure except for high concentrations, which lowered the NAD⁺ levels in L4 worms 24 h post-treatment. Interestingly, however, mutant worms lacking components of NAD⁺-dependent pathways involved in genome maintenance, namely pme-2, parg-2, and sirt-2.1 showed a higher and compound-specific susceptibility, indicating an active role of NAD⁺ in genotoxic stress response. In conclusion, the present results demonstrate that C. elegans represents an attractive model to study the toxicology of alkylating agents, which supports its use in mechanistic as well as intervention studies with major strength in the possibility to analyze toxicities at different life cycle stages.

Keywords: C. elegans; NAD+; alkylating agents; life cycle toxicities; mustards; neurotoxicity.

Figure 1

Lethality of C. elegans larvae L1 and L4 after CEES or HN2 exposure (a,b). Synchronized WT (N2) worms were exposed to alkylating agents for 30 min in M9. Next, approximately 30 worms were transferred onto 35 mm NGM plates in technical triplicates; 24 h later, dead and alive worms were scored, and the percentages of alive worms were normalized to solvent control (0 mM). Results were expressed as mean ± SEM (n = 3 biological replicates). For statistical analysis, two-way ANOVA was performed; ** p < 0.01, *** p < 0.001 vs. L1.

Figure 2

Lethality of adult C. elegans after CEES or HN2 exposure (a,b). Synchronized WT (N2) worms were cultivated on NGM + FUdR until adulthood day 1 (A1), day 5 (A5), or day 10 (A10), when worms were collected from plates and exposed to alkylating agents for 30 min in M9. Next, approximately 30 worms were transferred onto 35 mm NGM + FUdR plates in technical triplicate and incubated at 20 °C; 24 h later, dead and alive worms were scored, and the percentages of alive worms were normalized to solvent control (0 mM). Results were expressed as mean ± SEM (n = 3 biological replicates). For statistical analysis, two-way ANOVA was performed; ** p < 0.01, *** p < 0.001 vs. A1, # p < 0.05 vs. A5.

Figure 3

Germline apoptosis after CEES or HN2 exposure. Synchronized ced-1p::GFP (MD701) worms at the L4 stage were exposed to CEES or HN2 for 30 min in M9. Next, worms were placed on NGM plates and incubated at RT; 24 h later, adult hermaphrodites (at least 25 per sample) were manually picked and examined under a fluorescence microscope. The number of apoptotic germ cells (GFP-positive) observed in the gonad death zone loop (red triangles) was scored, and means for each sample were calculated. The scale bar represents 100 µm (a). Results were expressed as mean ± SEM ((b), n = 3 biological replicates). For statistical analysis, unpaired two-tailed t-tests were performed; * p < 0.05, ** p < 0.01 vs. solvent control (0 mM).

Figure 4

C. elegans offspring number and embryonic lethality after CEES or HN2 exposure. Synchronized WT (N2) L4 worms were exposed to CEES or HN2 for 30 min in M9. Next, worms were placed on NGM plates and incubated at 20 °C; 24 h later, adult hermaphrodites were manually picked and placed individually (technical quadruplicates) on 35 mm NGM plates and incubated at 20 °C for 24 h. For each hermaphrodite, the number of offspring during the first 24 h of adulthood was scored. Means for each sample were normalized to control (M9) ((a), n = 3 biological replicates). Additionally, the number of eggs unhatched during the following 24 h was scored and normalized to the offspring number ((b), n = 3 biological replicates). Results were expressed as mean ± SEM. For statistical analysis, a one-way ANOVA with Dunnett’s multiple comparisons test was performed; * p < 0.05, ** p < 0.01, *** p < 0.001 vs. solvent control (0 mM).

Figure 5

C. elegans dopaminergic neurons toxicity after CEES or HN2 exposure. Synchronized dat-1p::GFP (BY200) L4 worms were exposed to CEES or HN2 for 30 min in M9. Next, worms were placed on NGM plates and incubated at 20 °C for 24 h. Adult (A1) worms were collected and analysed using a worm sorter for the decrease in fluorescence intensity ((a), n = 4 biological replicates). The survival of BY200 was also determined: after treatments, worms were transferred onto 35 mm NGM plates in technical triplicates; 24 h later, dead and alive worms were scored, and the percentages of alive worms were calculated for each sample ((b), n = 3 biological replicates). Representative photos of dopaminergic neurons in the head area were taken with the confocal microscope; the scale bar represents 50 µm (c). Data are expressed as mean ± SEM. For statistical analysis, unpaired two-tailed t-tests were performed; * p < 0.05, ** p < 0.01 vs. solvent control (0 mM).

Figure 6

NAD⁺ levels in C. elegans after CEES or HN2 exposure. Synchronized WT (N2) worms at different life cycle stages: larvae L4 ((a,b); n = 1–3 biological replicates), adulthood day 1 (A1; (c,d); n = 2–3 biological replicates), day 5 (A5; (e,f); n = 2–3 biological replicates), or day 10 (A10; (g,h); n = 2–3 biological replicates) were exposed to CEES or HN2 for 30 min in M9. NAD⁺ was extracted from whole worms immediately (0 h) or after incubation for 2 h, 4 h, or 24 h at 20 °C on NGM + FUdR plates. NAD⁺ levels were measured with a cycling assay and normalised to the total protein levels measured with a BCA assay. Results were normalised to the value of the control sample (M9) and expressed as mean ± SEM. For statistical analysis, two-way ANOVA with Dunnett’s multiple comparisons test was performed, * p < 0.05, ** p < 0.01, vs. solvent control (0 mM).

Figure 7

Lethality of C. elegans with deficient NAD⁺-dependent system after CEES or HN2 exposure (a,b). Synchronized WT (N2) and mutant worms: pme-1(ok988) (RB1042), pme-2(ok344) (VC1171), parg-1(gk120) (VC130), parg-2(ok980) (VC641), sirt-2.1(ok434) (VC199), sirt-2.3(ok444) (RB654) were cultivated on NGM until adulthood day 1 (A1), when worms were exposed to alkylating agents for 30 min in M9. Next, approximately 30 worms were transferred onto 35 mm NGM plates in technical triplicates and incubated at 20 °C; 24 h later, dead and alive adult worms were scored, and the percentages of alive worms were normalized to solvent control (0 mM). Results were expressed as mean ± SEM (n = 3 biological replicates). For statistical analysis, two-way ANOVA was performed; * p < 0.05, ** p < 0.01 vs. WT.