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 (24 h) 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.

1. Introduction

Nicotine, a major component of tobacco products, is one of the most abused substances that lead to addiction. Nicotine targets nicotinic acetylcholine receptors (nAChRs), stimulates the mesolimbic dopamine system, and results in dependence [1,2]. Chronic exposure to nicotine in heavy smokers or via second hand smoking is a major component for developing addiction behaviors. It is known that long-term exposure to nicotine causes overexpression of nAChRs, especially the heteromeric receptors, contributing to the adaptive mechanism essential for drug-dependence and addiction [3]. Long-term smoking is also associated with various chronic disorders including several cancer types, cardiovascular diseases, and chronic obstructive pulmonary disease (COPD). These may be attributed to the cumulative oxidative stress, DNA damage, fatty acid buildup, and inflammatory status [4,5]. In addition, studies have noted a positive relationship between increased mortality rate and prenatal nicotine exposure [6]. As a result, cigarette smoking is one of the most important causes of premature death and disability world-wide [7]. Thus, investigating mechanisms of nicotine effects and seeking novel biochemical targets has long been an interest of drug industry, in an effort to develop early intervention strategies.

The model organism Caenorhabditis elegans has been widely used to decipher mechanisms of action of various abused substances including nicotine. Compared with other animal model systems, C. elegans has many unique advantages, including a completely decoded genome, short life cycle, and large brood size. The C. elegans genome encodes 28 nAChR genes and over one-third of the total 302 neurons in an adult hermaphrodite are involved in cholinergic transmission, which is important for regulating various drug effects [8,9]. Besides the nAChR genes, approximately 60–80% of C. elegans’ genes are conserved with humans [10]. In addition, the reproductive process of gametogenesis has been clearly demonstrated in C. elegans, make it an ideal model system to study reproductive effects of toxicants/drugs. All these advantages facilitate the study of the genetic basis of nicotine toxicity in vivo.

It is known that the nAChR agonist nicotine stimulates body wall muscle contraction and promote egg-laying behaviors in C. elegans [11]. Acute exposure to nicotine at high levels causes paralysis of the body wall muscle, while chronic exposure to high nicotine dosage results in alterations of egg-laying behaviors. C. elegans wild-type (wt) animals lay eggs in a temporal pattern: egg-laying tends to be clustered in short clustered episodes, which are separated by longer intervals during which eggs are not laid [12]. Chronic exposure to a relatively high concentration of nicotine (30 mM) results in time shortening of the egg-laying events and elongation of the intercluster time interval [13,14]. Feng et al. firstly defined nicotine-dependent behaviors following acute and chronic exposure (16 h) to low levels of nicotine (0.5–5 μM) [9]. Another study revealed that nicotine exposure induces concentration-dependent (at a dose range of 0.001–30 mM) and time-dependent (from 0 min to 300 min) changes in locomotion behaviors [15]. However, effects of chronic nicotine exposure on egg-laying behaviors and potential impacts on reproduction remain largely unknown. The C. elegans egg-laying circuit consists of at least three types of cells: the vm2 vulval muscles, and the hermaphroditespecific HSN and VC motorneurons (reviewed by [16]). The muscle cells receive synaptic input from neurons through the action of various neurotransmitters (mainly acetylcholine, serotonin, and neuropeptides) on corresponding membrane receptors. Although researches have identified many genes that play important roles in egg-laying and reproductive aspects, few studies have worked on the nicotine-induced gene expression pattern, specifically on identifying key gene players responding to chronic low nicotine exposures.

In this study, we employed C. elegans as a model organism to investigate the effect of chronic low nicotine exposure on the response to stimulus, reproduction, and gene expression of eighteen selected genes implicated in egg-laying, locomotion, and stress-response.

2. Materials and methods

2.1. C. elegans culture and treatment

Wild-type C. elegans strain Bristol N2 were grown in Petri dishes on 6-cm nematode growth medium (NGM) and fed with Escherichia coli strain OP50 according to a standard protocol [17]. Worms at larval stage 3 (L3) from an age-synchronized culture were used in all experiments. Age-synchronized cultures were obtained according to a previous report [18]. Worms were then placed on an NGM plate to allow hatching with no food for an arresting period to ensure all worms were closely synchronized. The worms were then transferred to OP50 seeded NGM agar plates, allowed developing into L3 stage (32–36 h after L1), and then collected for nicotine dosing.

l-Nicotine (98% pure) was obtained from Fisher Scientific. Different concentrations of nicotine dosing solution were made in K-medium (0.032 M KCl and 0.051 M NaCl): 1 ppm, 3.16 ppm, 10 ppm, and 31.6 ppm, corresponding to 6.17, 19.5, 61.7, and 194.5 μM, respectively. K-medium was used as a control. L3 stage worms were exposed to each of five different dosages for 24 h with E. coli OP50 as food.

2.2. C. elegans recovery from dosing

After 24 h of exposure, worms were collected, rinsed, and transferred to 12 well tissue culture plates filled with OP50-seeded NGM agar. The worms were allowed to acclimate for 1 h and then observed for general morphology and movement using a dissecting microscope. If a worm was not moving it was touched with a platinum wire. Worms not responding to gentle platinum wire stimulation were counted as failure to recovery from dosing. Around 50 worms were placed in each well in triplicate for one biological replicate. Four biological replicates (four independent experiments) were performed for totaling ~600 worms per dose.

2.3. Effect of nicotine exposure on C. elegans 24-h egg-laying pattern

After 24 h of nicotine exposure, worms were collected, rinsed, and individually placed into 12-well OP50-seeded NGM plates for egg-laying. Eggs and hatched larvae were counted every 24 h for 72 h post-dosing. Three independent experiments were performed with three replicates per dosage each experiment, totaling nine replicates per dosed group for statistical purpose.

2.4. Effect of nicotine exposure on protein-coding gene expression in C. elegans

After 24 h of nicotine exposure, worms were collected and rinsed with M9 buffer 3–4 times to remove residual nicotine and bacteria. Worms were frozen in liquid nitrogen and then stored in at −80 °C until RNA extraction.

Total RNA was isolated using the TRI Reagent® Reagent from Applied Biosystems according to the manufacturer's protocol. Total RNA was then quantified and assessed for quality using a Nanodrop ND-1000 (Nanodrop Technologies). Eighteen protein-coding genes (cat-10, egl-10, egl-19, egl-44, elg-47, egl-5, hlh-14, lin-39, ric-3, unc-103, unc-43, unc-50, unc-68, sod-1, pink-1, oxi-1, age-1, and old-1) that relate to worm reproductive traits and stress response were selected to test the impact of nicotine exposure on gene expression.

Applied Biosystems TaqMan microRNA Assays were employed to detect and quantify C. elegans protein-coding genes using quantitative real time PCR (qRT-PCR) according to the manufacturer's instructions. There were two steps in the TaqMan miRNA Assays: (a) reverse transcription of mRNA to a cDNA sequence using a poly(T) primer for protein-coding gene, and (b) quantitative real-time PCR. In short, single-stranded cDNA was generated from 500 ng of the total RNA from control, 1 ppm, 3.16 ppm, 10 ppm, and 31.6 ppm nicotine exposed C. elegans samples. Reverse transcription was carried out in a15 μL solution, which contained 500 ng of total RNA, 1 mM each of dNTPs, 1 μL MultiScribe Reverse Transcriptase (50 U/μL), 1.5 μL 10× RT Buffer, 0.188 μL RNase Inhibitor, and 3 μL 5× Taqman RT primer. The reverse transcription reaction was performed with an Eppendorf Mastercycler Personal PCR machine (Westbury, NY) with the following temperature program: initial 16 °C for 30 min followed by 42 °C for 30 min; then, the reaction was held for 5 min at 85 °C; finally held at 4 °C until next analysis or stored at −20 °C. For qRT-PCR analysis, each reaction was performed in a 20 μL solution, which contained 2 μL of RTPCR product (10-fold dilution from RT-PCR), 10 μL of SYBR green PCR Master Mix, 2 μL of forward and reverse primer and 6 μL of DNase/RNase free water. The temperature program for qRT-PCRs was 95 °C for 10 min followed by 40 cycles of 95 °C for 15 s and 60 °C for 60 s.

All reactions had three technical replicates and each dose had three biological replicates. Supplemental Table 1 shows the primer information of the 18 tested genes. In qRT-PCR, Y45F10D, a conserved iron binding related protein was employed as an endogenous reference gene for normalizing qRT-PCR results [19]. Relative gene expression was analyzed using the ΔΔCt method [20].

Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.reprotox.2013.05.007.

2.5. Statistical analysis

Statistical differences between the control and exposed worms were determined using standard statistical software (SPSS). Analysis of variance (ANOVA) was used for comparing means of different treatment groups. For time-series data obtained from egg-laying assays, repeated measures ANOVA using time as the factor were employed to test differences among treatment groups. If there was a significant difference among treatment groups at p < 0.05 level, least significant difference (LSD) multiple comparisons were conducted to compare means of each group.

3. Results

3.1. C. elegans recovery from dosing

The recovery rate of C. elegans was significantly affected by nicotine exposure (p = 0.031). After 24 h of exposure and a 1 h acclimation period, the percentage of C. elegans recovery decreased as the dose increased. The “No response to touch” rate was increased as nicotine concentration increased (Fig. 1). Nicotine concentrations equal to and larger than 3.16 ppm (19.5 μM) significantly affect the recovery rate as compared to control. There was a dose-dependent increase in the percentage of animals that failed to respond to touch stimulus after dosing. From low to high concentrations, there were four distinct statistical groups defined including control (represented as a, ab, b, c and d on Fig. 1).

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.

3.2. Effect of nicotine exposure on C. elegans 24-h egg-laying pattern

We did not observe eggs or hatched larvae in the dosing solution during nicotine exposure. During the first 24 h post-dosing, there was no significant difference in egg production among control and treatment groups. The average numbers of eggs laid (counted as eggs plus hatched larvae) during the first 24 h post-dosing for control and treatments were around 73 (Fig. 2). More eggs were laid during the second 24-h period (24–48 h post-dosing) (Fig. 2); the average numbers of eggs laid for control and treatment groups were around 112. The 10 ppm dosed group produced more eggs laid compared to the control; with a 10% increase (p < 0.05) during the second 24-h period. Other treatments did not significantly affect the egg production during this period. The number of eggs laid during the third 24-h period (48–72 h post-dosing) dropped, the average numbers of eggs laid for control and treatment groups were around 84 (Fig. 2). The highest treatment group (31.6 ppm) produced ~6% more eggs laid than control and the increase was significant (p < 0.05). Other treatments did not affect the egg production during this period. Fig. 3 shows the accumulated total of eggs laid over time. No significant difference observed among the two low nicotine treatment groups (1 ppm and 3.16 ppm) and control, however high concentrations (10 and 31.6 ppm) of nicotine caused worms to produce significantly more eggs after 48 and 72 h post-dosing (Fig. 3). At 72 h post-dosing, the control group produced 261 eggs per worm while worms exposed to 1, 3.16, 10 and 31.6 ppm nicotine produced 265 (p = 0.677), 265 (p = 0.645), 276 (p = 0.009), and 277 (p = 0.009) eggs, respectively. About 4% more eggs were produced in the 10 ppm and 31.6 ppm groups during the 72 h period after nicotine exposure.

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.

3.3. Effect of nicotine exposure on protein-coding gene in C. elegans

This study identified novel gene targets of nicotine by using a candidate gene approach; 18 candidate genes were selected for testing, including cat-4, egl-10, egl-19, egl-44, elg-47, egl-5, hlh-14, lin-39, ric-3, unc-103, unc-43, unc-50, unc-68, sod-1, pink-1, oxi-1, age-1, and old-1. Table 1 shows the functional classification of tested genes. Chronic exposure to nicotine at the micromolar range induced aberrant gene expressions in wt C. elegans across all dosed concentrations. Fig. 4 shows the fold change in gene expression of the 18 tested genes, varying from 12.5-fold down-regulation (old-1) to 138.1-fold up-regulation (hlh-14). Statistical analysis indicated that there were 10 tested genes expressed aberrantly in response to at least one dosage of nicotine. These 10 genes are egl-10, egl-44, hlh-14, ric-3, unc-103, unc-50, unc-68, sod-1, oxi-1, and old-1. Among the 10 genes, 8 genes (egl-10, egl-44, hlh-14, ric-3, unc-103, unc-50, unc-68, and oxi-1) were significantly up-regulated, 2 genes (sod-1 and old-1) were significantly down-regulated. Gene regulation is the most active at two low nicotine treatment groups; 3 (egl-44, ric-3, and old-1) and 7 tested genes (egl-10, hlh-14, unc-103, unc-50, unc-68, sod-1, and oxi-1) were differentially expressed at 1 ppm and 3.16 ppm treatment groups, respectively. In contrast, only 3 (egl-44, ric-3, and old-1) and 1 tested genes (ric-3) were significantly changed at 10 ppm and 31.6 ppm treatment groups, respectively. Changes in gene expressions were the most significant under 3.16 ppm nicotine exposure; six genes (egl-10, hlh-14, unc-103, unc-50, unc-68, and oxi-1) displayed at least 10-fold significant up-regulation.

Table 1.

Functional classification of the eighteen tested genes.

Gene symbol	Locus tag	Gene description	Egg-laying/reproduction	Develop/cell fate	Muscle and neuron	Stress response/signaling	Life span	References
cat-4	F32G8.6	abnormal CATecholamine distribution	√		√			[37]
egl-10	F28C1.2	Egg laying defective	√		√	√		[38–40]
egl-19	C48A7.1	Egg Laying defective	√		√			[41–43]
egl-44	F28B12.2	Egg Laying defective	√	√				[25,44]
egl-47	C50H2.2	Egg Laying defective	√		√			[45]
egl-5	C08C3.1	Egg Laying defective	√	√		√		[46–50]
hlh-14	C18A3.8	Helix Loop Helix	√	√				[29,51]
lin-39	C07H6.7	abnormal cell LINeage	√	√				[52–55]
ric-3	T14A8.1	Resistance to Inhibitors of Cholinesterase			√	√		[32,56–58]
unc-103	C30D11.1	UNCoordinated	√		√			[30,59]
unc-43	K11E8.1	UNCoordinated	√	√	√		√	[60–62]
unc-50	T07A5.2	UNCoordinated			√	√		[31,63]
unc-68	K11C4.5	UNCoordinated			√			[34,35,64]
sod-1	C15F1.7	SOD (superoxide dismutase)	√			√	√	[58,65,66]
pink-1	EEED8.9	PINK (PTEN-INduced Kinase) homolog			√	√		[67]
oxi-1	Y39A1C.2	Oxidative stress Induced				√		[24,68]
age-1	B0334.8	AGEing alteration		√			√	[69,70]
old-1	C08H9.5	Overexpression Longevity Determinant				√	√	[71,72]

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).

4. Discussion

Acute nicotine exposure at high concentrations (20–30 mM) causes rapid paralysis of C. elegans body wall muscle [13]. Since nicotine exposure in humans also occurs at a low dosage range and last for a prolonged period of time, it is important to investigate effects of chronic exposure to low concentrations of nicotine. Human nicotine blood concentration reaches ~500 nM following the consumption of one cigarette [21]. In habitual smokers, the blood nicotine level ranges from ~30 to ~300 μM [22]. In C. elegans, although nicotine-dependent behaviors were observed following chronic exposure (16 h) to low nicotine levels (500 nM–5 μM)[9], the characterization of genetic changes following exposure has been less completed.

In this study we observed a dose-dependent loss-of-response to stimuli in C. elegans exposed to low concentrations (micromolar range) of nicotine for 24 h in liquid medium. Worms that failed to respond appeared rigid, straight or exhibited a boomerang-shaped body line, suggesting chronic nicotine exposure may cause paralysis even at micromolar concentrations. This may due to the increased cuticle permeability under chronic exposure and during molting between larval stages.

It is known that nicotine exposure affects egg-laying behaviors [13,14]. However, it is not clear that if nicotine affects eggs production. At 20 °C, C. elegans begins to lay eggs as they become young adults (~65 h after hatching) and the total egg-laying event lasts for about 2.5 days, ending ~128 h post-hatching [23]. As most eggs are produced during the first 72 h of adulthood, we used the first 3-day egg productions as a more sensitive reproduction effect for comparisons. We found that during the 24–48 h period after exposure, both control and nicotine-treated C. elegans produce significantly more eggs than the first and the third 24 h period. This is consistent with the fact that the egg-laying maximal of wt C. elegans occurs ~31 h after the egg-laying begins [23]. Chronic exposure to 10 ppm nicotine seems to promote earlier egg-laying; as during the second 24 h period, 10 ppm group worms laid ~10% more eggs than others (p < 0.05). By 72 h post-dosing, worms treated with 10 ppm (61.7 μM), and 31.6 ppm (194.5 μM) nicotine slightly promoted egg production by ~4% compared to the control (p < 0.05), however this is unlikely to be biologically significant. Although the entire brood sizes were not compared, up to one-fifth of eggs can be produced after 72 h of adulthood. Taken together, chronic exposure to micromolar concentrations of nicotine promotes early egg-laying events and slightly increases eggs production during the first 72 h of adulthood.

We also investigated the expression patterns of 18 protein-coding genes belonging to different gene families. While many of these selected genes are known to regulate functions including egg-laying and stress response [11,24,25], little is known whether these genes are targets of nicotine and how nicotine affects expressions of these gene. For example, egl family genes egl-10 and egl-44 exhibited significant changes. Most egl genes were identified in screening of egg-laying defective mutants; their functions are associated with the formation of transcription factors, transmembrane G-protein-coupled receptors, and modulating motor neuron functions related to egg-laying [25,26]. We found that at 1 ppm nicotine, egl-44 exhibited a significant 5.6-fold up-regulation, suggesting egl-44 are among those early response genes sensitive to low dose nicotine exposure.

Egl-10 is highly conserved with mammalian regulator of G protein signaling (RGS) genes, which regulate G-protein signaling pathways and modulate frequencies of periodic behaviors. While loss-of-function mutant, overexpression of egl-10, and wild-type all produce similar number of eggs, the egl-10 overexpressers laid eggs more frequently than the wild-type [27]. Although C. elegans egg-laying is a type of periodic behavior, the regulation of egl-10 by nicotine has never been reported before. The eggs of C. elegans are laid in clustered episodes with intervals between the events [12]. It was reported that chronic exposure to 30 mM of nicotine for 16 h in NGM medium results in abnormality of this periodic behavior; the egg-laying cluster is shortened and the intercluster time interval is longer [13,14]. In this study we found that egl-10 exhibited 22.7-fold up-regulation under 3.16 ppm (19.5 μM) nicotine exposure. This suggests that egl-10 may play a key role in regulating the periodic egg-laying behaviors in response to nicotine exposure.

The other gene, egl-44, is mostly upregulated. Egl-44 encodes a putative transcription regulatory protein similar to the transcription enhancer factor (TEF) protein in vertebrates. It functions to promote the differentiation and migration of HSN motor neurons needed for egg-laying [25] and to prevent excess dendritic branching of the PVD neurons [28]. The hlh-14 also regulates differentiations and functions of HSN [29]. Hlh-14 was the most upregulated gene detected in this study under chronic treatment of 3.16 ppm nicotine. Taken together, the nicotine-induced aberrant expressions of egl-10, egl-44, and hlh-14 indicates an adaptive response to maintain the normal egg-laying activities during low nicotine exposure.

Given the primary target of nicotine is the nicotinic acetylcholine receptor (nAChR), it is not surprising to see that several cholinergic signaling-related genes were changed by chronic low nicotine exposure. The K⁺ channel gene unc-103 expresses in HSN neurons and vulval muscles, regulating acetylcholine-mediated muscle excitability and egg-laying behaviors. The loss-of-function unc-103 mutant hermaphrodites reduced egg-laying behaviors and laid embryos prematurely, while a supply of unc-103 mRNA to vulval muscle restored the egg-laying inhibition effects in M9 buffer [30]. The gene unc-50 encodes a Golgi protein required for traffic-king of assembled nAChRs to the plasma membrane in C. elegans [31]. In addition, the nicotine-induced gene ric-3 is also involved in the trafficking and assembly of several nicotinic acetylcholine receptors [32,33]. Interestingly, unc-50 and ric-3 exhibited similar trend in gene expression in this study, suggesting the trafficking and assembling of functional nicotinic receptor were subjected to regulation by nicotine exposure. The ryanodine receptor gene unc-68 regulates the coupling of excitation–contraction of body-wall muscle, required for normal locomotive behaviors [34,35]. All three unc genes and ric-3 are up-regulated >10-fold at the 3.16 ppm dose, suggesting active regulation of excitatory neuronal functions by the AChR agonist nicotine.

As nicotine induces oxidative stress and DNA damage in mammals [4,36], we tested if the oxidative stress-responsive genes are changed in C. elegans following nicotine exposure. We found that oxi-1 was significantly up-regulated while sod-1 and old-1 were in general down-regulated. This indicated nicotine may affect the stress-defense system itself, besides causing oxidative stress. Oxi-1 encodes a family of proteins homologous to human ubiquitin-protein ligase [24], suggesting the level ubiquitination may be increased under nicotine exposure. On the other hand, the down-regulation of sod-1 and old-1 induced by nicotine may result in more oxidative injuries and life-span defects in exposed animals. More studies need to be performed to investigate nicotine-related oxidative stress and associated DNA damage in C. elegans.

Although previous studies have investigated functions of many tested genes, this study identified genes affected by chronic micromolar range nicotine exposure. Some genes were identified as novel targets of nicotine that are important players involved in neuronal development, egg-laying, nAChR functions, oxidative stress and aging. Some of the nicotine-induced genes have orthologs in vertebrates and humans, including egl-10, egl-44, unc-103, unc-50, unc-68, oxi-1, and old-1. Novel gene targets identified in this study may provide new mechanic insights into nicotine toxicity in humans.