Impacts of chronic low-level nicotine exposure on Caenorhabditis elegans reproduction: identification of novel gene targets

Abstract

Effects and mechanisms of chronic exposure to low levels of nicotine is an area fundamentally important however less investigated. We employed the model organism Caenorhabditis elegans to investigate potential impacts of chronic (24h) and low nicotine exposure (6.17-194.5 μM) on stimulus-response, reproduction, and gene expressions. Nicotine significantly affects the organism's response to touch stimulus (p=0.031), which follows a dose-dependent pattern. Chronic nicotine exposure promotes early egg-laying events and slightly increased egg productions during the first 72 h of adulthood. The expressions of 10 (egl-10, egl-44, hlh-14, ric-3, unc-103, unc-50, unc-68, sod-1, oxi-1, and old-1) out of 18 selected genes were affected significantly. Other tested genes were cat-4, egl-19, egl-47, egl-5, lin-39, unc-43, pink-1, and age-1. Changes in gene expression were more evident at low dosages than at relatively high levels. Genes implicated in reproduction, cholinergic signaling, and stress response were regulated by nicotine, suggesting widespread physiological impacts of nicotine.

Keywords: C. elegans; Chronic exposure; Gene expression; Nicotine; Reproduction; Stress-response.

Fig. 1

Percentage no response to touch versus nicotine dosage. L3 C. elegans were dosed with different concentrations of nicotine in K-medium with food for 24 h and then subjected to gentle touch stimulus if not moving. Around 50 worms were placed in each well in triplicate for one biological replicate. Four biological replicates (four independent experiments) were performed totaling ~600 worms per dose. For each dose, the average “No response to touch”% of the 3 technical replicates was treated as one data point. This figure shows the mean and standard error (SE) of “No response to touch”% of 4 biological replicates (n = 4). Statistical analysis was performed by the ANOVA. Different letters indicated distinct statistical groups at p < 0.05 level.

Fig. 2

Effects of different doses of nicotine (ppm) on the 24-h egg-laying pattern over 72 h post-dosing. L3 C. elegans were dosed for 24 h and then placed individually in NGM agar well with OP50 to allow egg-laying. Eggs were counted at three time points: 24, 48, and 72 h after dosing. Data at the time point 24 h represent the number of egg laid during the period of 0–24 h post-dosing. Data at the time point 48 h represent the number of egg laid during the period of 24–48 h post-dosing. Data at the time point 72 h represent the number of egg laid during the period of 48–72 h post-dosing. Error bars indicate the standard error (SE). Different symbols of the same type represent different statistical groups comparing at the same time point by repeated measures ANOVA at p < 0.05 level.

Fig. 3

Effects of different doses of nicotine (ppm) on eggs production over 72 h post-dosing. L3 C. elegans were dosed for 24 h and then placed individually in NGM agar well with OP50 to allow egg-laying. Eggs were counted at three time points: 24, 48, and 72 h after dosing. Data at the time point 24 h represent the number of egg laid during the period of 0–24 h post-dosing. Data at the time point 48 h represent the number of egg laid during the period of 0–48 h post-dosing. Data at the time point 72 h represent the number of egg laid during the period of 0–72 h post-dosing. Error bars indicate the standard error (SE). Different symbols of the same type represent different statistical groups compared at the same time point by repeated measures ANOVA at p < 0.05 level.

Fig. 4

Expression fold change of the 18 protein-coding genes. L3 C. elegans were dosed for 24 h in K-medium and then RNAs were extracted for gene expression analysis. Fold changes of genes were normalized using Y45F10D.4 mRNA and are presented as relative units compared to control (control = 1, above 1 mean up-regulated, below 1 means down-regulated). Y-axis uses logarithmic scale and shows the mean and standard deviation (SD) of fold changes of 3 independent experiments for individual genes (n = 3).