A C. elegans model of electronic cigarette use: Physiological effects of e-liquids in nematodes

Strains

The following strains were utilized in this study: N2 Bristol (wild type) and CL2166 (Pgst-4::GFP::NLS). Strains CL2070 (Phsp-16.2::GFP; rol-6(su1006)), ZG120 (Pnhr-57::GFP; unc-119(+)), SJ4005 (Phsp-4::HSP-4::GFP), and SJ4058 (Phsp-60::GFP) were also tested but data from these strains is not included in the manuscript. All of these strains were obtained from the Caenorhabditis Genetics Center (CGC) at the University of Minnesota (Duluth, Minnesota, USA).

Nematode culture

Wild type (N2 Bristol) C. elegans nematodes were cultured according to standard methods [17]. The nematodes were maintained at 20 °C on nematode growth medium (NGM) agar plates seeded with an auxotroph strain of E. coli (OP50), and were routinely propagated to prevent starvation.

Nematodes were exposed to the e-cig liquids (V2 Platinum E-Liquid, V2CIGS/VMR Products LLC., Miami, USA) via cultivation on NGM agar plates containing a 1:500 dilution of the commercially available cartridge refill solution. The composition of each solution can be found on the V2 website at http://www.v2cigs.com/templates/v2v3/Download/ingredients_V2.pdf. In general, these solutions are comprised of a solvent base PG, which is generally ~70 % of the solution, and are supplemented with nicotine. The e-liquids used here contained either 0 or 2.4 % nicotine, and hence following dilution the final concentration of PG in the plates was 0.14 % and that of nicotine was either 0 or 48 ppm. Negative unsupplemented controls contained a 1:500 dilution of M9 buffer. Propylene glycol (1, 2-Propanediol), nicotine ((−)-Nicotine hydrogen tartrate salt), and paraquat (N,N’-dimethyl-4,4’-bipyridinium dichloride) were purchased from Sigma-Aldrich (Saint-Louis, MO, USA)

Production of vapor distillates

E-liquid vapors were produced by an iPV2S 7-70 W Box Mod (Green Leaf Technology Co., Ltd, Shenzhen, China) connected to a Nautilus Mini BVC Tank (Aspire, Shenzhen, China). A silicon tube was connected to the mouthpiece of the Nautilus Mini on one end, and the other end was placed in the lower part of a 50 ml conical tube, in which the condensation took place. The silicone tubing passed through a hole that was drilled in the lid of the conical tube, fastened to the edges of the hole with hot glue, and a layer of epoxy resin was applied on top of the dried hot glue to hold the tube in its place. The conical tube was suspended above liquid nitrogen inside a thermal container. Another tube passed through a second hole in the lid, and linked the tube to a capped 250 ml vented Erlenmeyer flask. The vent of this flask was connected through another silicone tube to the hub of a 60 ml syringe, which was used to draw in air through the vaping device. 750 μl of e-liquid or control was pipetted into the glass tank of the Nautilus Mini, and the device was activated for periods of 4 seconds with at least ten seconds between activation periods, both to simulate puffs as well as to avoid burning the wicking material within the atomizer. This was done several times to acquire a sufficient amount of the condensate. The Nautilus Mini was cleaned with water and the atomizer replaced between different treatments, and the tubes were cleaned with ethanol. The vaporized, distilled e-liquids were added to NGM plates in the same 1:500 dilution as the original e-liquids. The concentration of PG was presumed to be conserved and the concentration of nicotine was not significantly different from that in the original e-liquid, as determined by quantitative HPLC analysis (see below).

Post-embryonic development assay

Synchronized embryos were prepared using a standard method of alkaline bleach treatment [17] and placed on seeded NGM agar plates containing the diluted e-liquids. Larval nematodes were imaged at 24 h intervals, beginning approximately 12 h following hatching and continuing for 3 days, using a PixeLINK PL-B621MU-KIT microscopy camera (PixeLINK, Ottawa, Ontario) attached to an Olympus MVX10 stereomicroscope (Olympus, Japan). The length of the nematodes was calculated using ImageJ 1.49 h software (Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA, http://rsb.info.nih.gov/ij/).

Adult nematode lifespan assay

Worms were synchronized as described above using an alkaline bleach treatment to isolate embryos and cultured on e-liquid NGM agar plates for 3 days. 40 young adult nematodes were moved from each treatment plate to another plate that contained the same e-liquid supplemented with 0.1 mM FUDR (5-Fluoro-2’-deoxyuridine, F0503, Sigma-Aldrich, Saint-Louis, MO, USA). FUDR prevents the development of offspring and permits the first generation nematodes to be unambiguously identified as they age. The plates were maintained at 20 °C, with worms being moved to new, identical plates at least once to prevent starvation, and survival was assessed daily under a SMZ800 stereomicroscope (Nikon, Japan). Once per day, nematodes that didn’t actively move backward after being touched were counted as dead. Nematodes that were found dead of obvious causes unrelated to age, such as vulval rupture, were censored instead of being counted as dead.

Brood size assay

Prior to the first eggs being laid by a synchronized population, several L4-young adult nematodes were individually moved onto new treatment plates where they were allowed to lay eggs for 24 h. The adult nematodes were then moved again onto separate plates at 24 h intervals over the course of 5 days. The plates were maintained at 20 °C, and the number of progeny was counted once they themselves had reached adulthood.

Thrashing assay

Young adult nematodes were individually transferred into a drop of liquid M9 with a volume of ~5 μl, which was placed on top of glass cover slide, and the worms were allowed 10–15 seconds to recuperate. The slide was placed under a standard dissecting microscope and video recordings were acquired for a period of 15 seconds. A discrete thrash was defined as a full change in the lateral bending direction of the whole nematode corpus. The number of thrashes performed by the nematodes during the recorded period was counted, and divided by the length of the recording to calculate the frequency of thrashing.

Fluorescent imaging

Images were obtained through the University of Rochester Light Microscopy Core on a FV1000 Olympus laser scanning confocal microscope with a 10x objective (Olympus, Japan) or using an epifluorescent rig consisting of a Nikon TE2000 inverted microscope (Nikon Instruments, Japan) equipped with a Cooke SensiCam cooled CCD and TILL monochromator (Till Photonics, Germany). All excitation-emission wavelengths and filters were appropriate for imaging GFP. Images were imported into ImageJ software (NIH) and average fluorescent intensities were calculated for each treatment (n = 10–14 worms and two experimental replicates).

Analytical determination of nicotine concentration and statistics

Nicotine concentrations in the e-liquids and vapor distillates were obtained via HPLC (Shimadzu, Japan), as described [18] using a Phenomenex Gemini C18 column.

Statistical analyses

A two-way ANOVA with Bonferroni post-hoc analysis was used to test significance. For lifespan analysis, the probability of survival was calculated and analyzed using Kaplan-Meier statistics. For HPLC analysis of nicotine content, a simple unpaired t-test was used.

The physiologic effects of e-liquids in the model organism C. elegans are mainly due to PG and nicotine

C. elegans are soil nematode and widely-used as a genetic model organism. Worms develop through four larval stages prior to entering reproductive adulthood, with a normal adult measuring ~1 mm in length (Fig. 1a). The consequence of exposure to e-cig juices (e-liquids) on development was determined by supplementing Nematode Growth Media (NGM) with solvents that included 1) M9 as a negative control, 2) purified PG, which typically comprises ~70 % volume of the e-liquid, or 3) commercially available V2 Platinum e-Liquid cartridge refills. The three e-liquid flavors tested were: V2 Red (“classic tobacco”), menthol, and grape. Collectively, these five variable additives are referred to hereafter as “solvent groups”.

Exposure of worms to environmental toxicants requires their absorption from the environment, and these toxicants must pass through the cuticle. Hence, IC50 can be higher in worms than in mammals [19]. Often this necessitates an empirical determination for effective dosage. Our criteria during this process was that we choose a low enough dose of e-liquid to avoid overt changes in behavior that are known to be driven by nicotinic receptors, such as egg-laying [20], but a high enough dose to observe a slight reduction in growth rate. Our preliminary results suggested that a 1:500 dilution met these criteria. This dilution corresponds to ~0.16 % PG in the NGM plates, and for e-liquids that contain 2.4 % nicotine, which is the highest concentration available in the V2 Platinum series, this equates to 48 ppm nicotine . Reference controls were supplemented with purified PG and nicotine obtained commercially. The plates were seeded with OP50 bacteria, the normal food source fed to worms in a laboratory setting, and worms were grown from synchronized embryos. Growth rates were calculated by measuring nematode length at 24-hour intervals over 3-days, corresponding to developmental times of approximately 12, 36 and 60 h (Fig. 1a). All treatments were performed in parallel, with 10–20 worms measured per treatment and three experimental replicates performed at different times

None of e-liquids exerted overtly malign effects, and the treated worms appeared morphologically normal (data not shown). As expected, there was a main effect of nicotine (Fig. 1b), but this effect was not dependent upon the solvent group. Unexpectedly, however, PG treated worms were smaller than the negative controls, independent of nicotine (Fig. 1b). This was also true for V2 Red. These results suggested that PG and nicotine are the main contributors to the observed alterations in size. The fact that these differences were generally mirrored at earlier time points (data not shown) suggests that this was an effect of growth rate rather than a reduction in terminal size.

Growth rates in C. elegans often reflect dietary intake and metabolism. Another stereotypical measure of metabolism is brood size. After reaching adulthood, hermaphrodite C. elegans begin to lay eggs, with a fertile period extending ~3 days and a normal brood size of ~250-300 progeny. In order to support this astounding fecundity, worms need to convert their body mass into embryos on a daily basis. Most of this energy conversion occurs in the intestine, which is the site of nutrient uptake, fat storage, and vitellogenin synthesis. Hence, brood size is tightly coupled to nutrient availability and has been used as a measure of metabolic normalcy. Dietary restriction as well as stress or genetic mutations in signaling pathways that respond to stress have all been shown to influence embryo output [21–23]. Here, we assessed the effect of e-liquids on total brood size, measuring the average from five worms per treatment, with three experimental replicates performed at different times. As was the case with developmental rate, the data in Fig. 1c suggest that brood size is also reduced by exposure to PG. The p-value for the other solvent groups was suggestive of an effect, but did not reach significance. Finally, as with development, there was a main effect of nicotine, but no interaction between nicotine and the solvent groups.

C. elegans have also been used extensively to study aging, and many types of stress have been shown to elicit changes in lifespan through specific genetic determinants (for review, see [9]). However, in our hands two independent analyses using 20–40 worms per treatment showed that the e-liquids did not significantly influence lifespan (data not shown). In contrast to lifespan, vitality or “graceful aging” refers to the rate at which age-related physiological changes occur during an organism’s lifespan. Vitality has traditionally been correlated with locomotory rates in worms, which tend to decrease with age. Here, we used thrashing assays to assess vitality, where worms are placed in a drop of liquid M9 buffer and their rate of body bends are calculated post-hoc via recordings obtained on a stereomicroscope. Recordings were made of young adult worms and compared to older adult worms as a function of e-liquid exposure throughout life. The data in Fig. 1d represents the average of three independent replicates of five individual worms per replicate. As with the other measures tested, there was a main effect of nicotine (Fig. 1d). The worms that were exposed to PG and V2 Red containing nicotine fared significantly worse than their counterparts exposed to nicotine alone (Fig. 1d). As expected, the thrashing rate was reduced in older worms, but there was no difference among solvent groups or as a function of nicotine (Fig. 1d).

PG and e-liquids elicit an SKN-1/Nrf-2 stress response independent of nicotine

C. elegans stress responses are well-conserved with mammals, particularly at the cell biological level, and are represented by canonical signaling pathways that have been extensively characterized through genetic approaches. Critical transcription factors have been identified in pathways that respond to various types of stress, including nutrient availability, such the FOXO ortholog DAF-16, which is the terminal effector of the worm insulin-like signaling cascade [24]; oxidative stress, such as the Nrf-2 ortholog SKN-1 [25]; hypoxia, such as HIF-1 [26]; and proteotoxicity, such as XBP-1 [27] and ATFS-1 [28], which act at the level of endoplasmic reticular and mitochondrial unfolded protein responses, respectively. One of the advantages of working with C. elegans is a wealth of genetic reagents. For the purposes of this work, those reagents included transgenic strains where the promoters from specific targets of each of these transcription factors, shown in Fig. 2, have been fused to the green fluorescent protein GFP. Hence, fluorescent output can be used as a surrogate for signal pathway induction.

Each of the five transgenic strains shown in Fig. 2 was screened for induction by either PG or the V2 Red e-liquid ± nicotine using fluorescent microscopy (e-liquids containing other flavorings were not tested). Of the five strains tested, only one exhibited visible activation: the detoxification/antioxidant response factor SKN-1. Expression of the SKN-1 target gene gst-4 was visible under basal conditions (Fig. 2b), but was greatly enhanced by exposure to 10 mM paraquat, a stereotypical oxidative stress widely used in worms (Fig. 2a). Expression was also increased, though less dramatically, in response to PG and V2 Red (Fig. 2c, d, f, and g). Induction was independent of nicotine (Fig. 2e). These data indicate that PG is sufficient to elicit a mild oxidative stress response in worms, while nicotine is not.

Distilled vapor extracts of e-liquids exert similar effects as e-liquids themselves

One of the concerns regarding e-liquid vapors is that the vaporization process may result in free radical formation, which could cause oxidative stress and ultimately result in tissue damage over time. Given our observation that the detoxification/antioxidant response factor SKN-1 target gene gst-4 is induced by e-liquids, we hypothesized that vaporization may exacerbate this responses. In order to expose worms to vaporized e-liquid, a distillation system was devised where an e-cig device was used to create a vapor that was then pulled through negative pressure into a flask suspended over liquid nitrogen (Fig. 3a). The parameters used for this distillation process mimicked those that a typical e-cig user would employ (Methods). HPLC analysis of nicotine content following distillation suggested that most of the vaporized nicotine was recovered (Fig. 3a). The distilled vapor extract was added to NGM plates at an identical dilution as the original e-liquid (1:500) and embryos were allowed to develop on these plates. While this model does not recapitulate the acute exposure to vapor experienced by an e-cig user, it did allow us to determine whether vaporization itself led to increased toxicity in the worm model. In this study, vapor extracts of PG were compared to the V2 Red e-liquid; the other flavorings menthol and grape were not tested. In addition, since e-cig devices are built to vape PG or glycerin based solvents, the negative control data, generated using an aqueous solvent, was not vaped and is replicated here from Fig. 1 strictly for comparison.

Somewhat surprisingly, most of the physiologic measurements, including body length, brood size, and thrashing rate, were similarly affected by treatment with the distilled vapor extracts as by the original e-liquids (Fig. 3b-d). However, several of these effects were suggestive but not significant. Nevertheless, there was no overt increase in toxicity following vaporization.

Finally, we observed that SKN-1 pathway induction of gst-4::GFP was higher in treated groups compared to controls, but was reduced compared to paraquat, suggesting that PG induces a sub-maximal stress response (Fig. 3e-j). It’s possible that PG triggers SKN-1 activity, which is essential for development of embryos and upregulates the expression of genes that result in modification, conjugation, and export of xenobiotics (for review, see [29]), in the context of detoxification rather than oxidative stress. However, we also recognize that free radicals are transient reactive molecules, and hence it’s also possible that the nature of the model, which does not permit exposure immediately upon e-liquid vaporization, may prevent us from observing their full effect.