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Toxicology and Applied Pharmacology

journal homepage: www.elsevier.com/locate/taap

Video-based kinetic analysis of calcification in live osteogenic humanembryonic stem cell cultures reveals the developmentally toxic effect ofSnus tobacco extract

Ivann K.C. Martineza,b, Nicole R.L. Sparksa,c, Joseph V. Madrida, Henry Affeldt IIIa,Madeline K.M. Veraa,c, Bir Bhanud, Nicole I. zur Niedena,b,c,⁎

a Department of Molecular, Cell & Systems Biology and Stem Cell Center, College of Natural and Agricultural Sciences, University of California Riverside, Riverside, CA92521, United Statesb IGERT Graduate Program in Video Bioinformatics and Cell, Molecular and Developmental Biology Graduate Program, University of California Riverside, Riverside, CA,United Statesc Environmental Toxicology Graduate Program, University of California Riverside, Riverside, CA, United Statesd Center for Research in Intelligent Systems, Bourns College of Engineering, University of California Riverside, Riverside, CA, United States

A R T I C L E I N F O

Keywords:Embryonic stem cellsCalcificationOsteogenesisDevelopmental toxicityTobaccoSnusTobacco-specific nitrosamineNicotineN′-nitrosonornicotine

A B S T R A C T

Epidemiological studies suggest tobacco consumption as a probable environmental factor for a variety of con-genital anomalies, including low bone mass and increased fracture risk. Despite intensive public health in-itiatives to publicize the detrimental effects of tobacco use during pregnancy, approximately 10–20% of womenin the United States still consume tobacco during pregnancy, some opting for so-called harm-reduction tobacco.These include Snus, a type of orally-consumed yet spit-free chewing tobacco, which is purported to expose usersto fewer harmful chemicals. Concerns remain from a developmental health perspective since Snus has not re-duced overall health risk to consumers and virtually nothing is known about whether skeletal problems fromintrauterine exposure arise in the embryo.

Utilizing a newly developed video-based calcification assay we determined that extracts from Snus tobaccohindered calcification of osteoblasts derived from pluripotent stem cells early on in their differentiation.Nicotine, a major component of tobacco products, had no measurable effect in the tested concentration range.However, through the extraction of video data, we determined that the tobacco-specific nitrosamine N′-ni-trosonornicotine caused a reduction in calcification with similar kinetics as the complete Snus extract. Frommeasurements of actual nitrosamine concentrations in Snus tobacco extract we furthermore conclude that N′-nitrosonornicotine has the potential to be a major trigger of developmental osteotoxicity caused by Snus tobacco.

1. Introduction

Aside from its well-known detrimental effects that include increasedcancer risk, heart disease and chronic obstructive pulmonary disease,tobacco use has been associated with increased risk of osteoporosis andfractures as well as delayed mineralization yield and kinetics duringbone healing (Godfrey et al., 2001; Parviainen et al., 2017; Seemanet al., 1983; Daniell, 1976; Cooper et al., 1988; Blum et al., 2002;Izumotani et al., 2003; Ortego-Centeno et al., 1997). In dental clinics,smoking is among the most common risk factors for post-tooth-extrac-tion complications (Ozkan et al., 2014). Experiments in mice provedthat smoke exposure negatively changed the material properties of bone

such that it is more prone to accumulate permanent damage (Seemanet al., 1983). Indeed, a large cohort study including 14,060 U.S. subjectsconcluded that smoking and bone mineral density were inversely cor-related (Benson & Shulman, 2005).

Exposure to tobacco smoke has long been known to increase themother's risk for spontaneous abortions and for premature delivery. Inthe 1980's it was concluded that cigarette smoke also caused manyabnormalities to embryos (Ejaz et al., 2009). Further, some laboratorystudies have begun to verify the detrimental effects of tobacco exposureto the embryonic skeleton (Paulson et al., 1988). Isolated epidemiolo-gical human studies have associated smoking while pregnant withlower neonatal bone mass (Godfrey et al., 2001) and prepubertal bone

https://doi.org/10.1016/j.taap.2018.11.006Received 22 September 2017; Received in revised form 13 November 2018; Accepted 16 November 2018

⁎ Corresponding author at: Department of Molecular, Cell & Systems Biology and Stem Cell Center, College of Natural and Agricultural Sciences, University ofCalifornia Riverside, Riverside, CA 92521, United States

E-mail address: nicolezn@ucr.edu (N.I. zur Nieden).

Toxicology and Applied Pharmacology 363 (2019) 111–121

Available online 20 November 20180041-008X/ © 2018 Elsevier Inc. All rights reserved.

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mass at age 8 (Jones et al., 1999; Micklesfield et al., 2006), although noincreased fracture occurrence was reported in children of smokingmothers (Jones et al., 2004; Jones et al., 2013; Hallal et al., 2009).However, more recent studies find a significant correlation between inutero tobacco exposure and childhood fracture risk (Parviainen et al.,2017) as well as neonate bone mineral quality (Godfrey et al., 2001).

Despite warnings, 10–12% of women in the U.S. consume tobaccoproducts during pregnancy (Tong et al., 2009), often using so-called‘harm-reduction' tobacco products, which are perceived as safer. Smo-keless tobacco, including Snus, falls in this category since it does notyield combustion products when used. Similar to chewing tobacco,consumption of Snus occurs orally: a small tobacco filled pouch isplaced between the upper lip and gums and the resulting juice isswallowed. In Nordic European countries, where Snus has been tradi-tionally consumed since the early 19th century, the prevalence of Snususe has increased in recent years as consumption of conventional ci-garettes has fallen (Pedersen & von Soest, 2014; Lund et al., 2014).North America has introduced Snus only in 2006, but market sales havealready doubled between 2009 and 2010, with Snus brands establishingthemselves among the top 10 moist snuff brands sold in the U.S. justtwo years after their introduction (Delnevo et al., 2014). Its colorfulpackaging and sweet flavoring especially markets Snus to youth, youngadolescents, and women. Indeed, the use of smokeless tobacco has in-creased among women of childbearing age both in Sweden and globally(Connolly & Alpert, 2008). Although Snus is marketed with claims ofreduced cancer risk, little is known about its potential harmful effectson developing bone cells.

To evaluate the risk of Snus exposure on the human skeleton, in thisstudy, we turn to human embryonic stem cells (ESCs). The differ-entiation of ESCs mimics the development of an embryo in vitro and hasbeen exploited for analysis of risk associated with xenobiotic exposurein many tissues, including bone (zur Nieden et al., 2010; zur Nieden &Baumgartner, 2010; Walker et al., 2014). The genesis of bone from ESCsor within the skeleton ends with the formation of hydroxyapatite fromcalcium and phosphate. This process of calcification is facilitated by theosteoblast, which secretes a collagenous matrix as the frame work forthe hydroxyapatite formation as well as all other extracellular matrix(ECM) proteins necessary for bone function (Boskey, 1996). Amongthese, osteocalcin (OCN) is uniquely found only in bone tissue, which iswhy its presence is often used as a biomarker for osteogenesis (Butteryet al., 2001; zur Nieden et al., 2003; Sottile et al., 2003; Ding et al.,2012; Rutledge et al., 2014; Sparks et al., 2018). The ability of osteo-blasts to calcify the ECM can also be confirmed by calcium specificstains, such as von Kossa or Alizarin Red (zur Nieden et al., 2003; Dinget al., 2012; Sparks et al., 2018; Puchtler et al., 1969; Rungby, 1993),but this type of assessment is often qualitative.

Quantitatively, absorbent calcium sensitive dyes, such as ArsenazoIII, can detect matrix-bound calcium (Davis et al., 2011). Becausequantification with calcium-sensitive dyes typically requires sacrificingthe culture, our lab has used the physical black appearance of the cal-cified matrix in still bright-field images to quantify the amount of cal-cium deposited by ESCs as they differentiate and when exposed to xe-nobiotics (zur Nieden et al., 2010; zur Nieden & Baumgartner, 2010;Walker et al., 2014; zur Nieden et al., 2007). The kinetic analysis of thissignature dark appearance of the calcified matrix as described in thispaper is the cornerstone in the assessment of Snus exposure on suchdeveloping cells. With it, we were able to determine the adverse effectsof Snus on differentiating osteoblasts, measurable already after 10 daysin culture. Furthermore, we were able to pinpoint the tobacco con-stituent N′-nitrosonornicotine as a potential driving force for the in-hibitory outcome of Snus exposure on osteogenesis.

2. Materials and methods

2.1. Cell culture

H9 human embryonic stem cells (hESCs) were obtained from WiCelland were cultured on Matrigel (BD Biosciences) treated culture plates inmTeSR 1 medium (Stem Cell Technologies) as feeder-free cultures at37 °C with 5% CO2 as described (Sparks et al., 2018). Colonies werepassaged every 5 days using accutase treatment (2–4min at roomtemperature) and a cell scraper to dislodge cell clumps from the plastic.Cells were used within passages 3–10 after thawing to prevent accu-mulation of karyotypic abnormalities and screened once a month formycoplasma contamination.

Human foreskin fibroblasts were provided by Dr. Derrick Rancourt(University of Calgary, Canada) and cultured in high glucose L-gluta-mine Dulbecco's modified Eagle's medium (DMEM, Corning) with 10%fetal bovine serum (FBS, Atlanta), 1% non-essential amino acids (NEAA,Gibco), and 0.5% penicillin/streptomycin (10,000 units/10,000 units,Gibco).

2.2. Osteogenic differentiation of embryonic stem cells

Differentiation was induced from confluent hESCs by the addition ofcontrol differentiation medium (CDM) composed of DMEM containing15% FBS (Atlanta Biologicals), 1% (v/v) non-essential amino acids,50 U/ml penicillin, 50 μg/ml streptomycin, and 0.1 mM β-mercap-toethanol (Sparks et al., 2018). Osteogenic differentiation mediumcomposed of CDM supplemented with 1.2× 10−7 M 1,25α(OH)2 Vi-tamin D3 (VD3; Calbiochem), 0.1 mM β-glycerophosphate (βGP), and20.8 μg/ml ascorbic acid (AA) was used from day 5 of differentiationonward (zur Nieden et al., 2003; Ding et al., 2012; Sparks et al., 2018).Non-osteogenic cultures were continuously cultured in CDM.

2.3. Immunocytochemistry

Cells were rinsed with phosphate buffered saline (PBS) and fixed in4% paraformaldehyde at 4 °C for 1 h. Fixed cells were permeabilizedwith 0.1% Triton X-100/PBS and stained with anti-OCN (AbCam;AB1857) in 4% FBS/PBS overnight at 4 °C. A secondary anti-rabbit IgGconjugated to Alexa Fluor 546 (Invitrogen; A10040) in 10% FBS/PBSwas incubated for 2 h at room temperature. Cultures were counter-stained with 4′,6-diamidino-2-phenylindole (DAPI) and visualized usinga Nikon Eclipse Ti microscope.

2.4. Cytochemical staining

Fixed cells were stained with 2% (w/v) Alizarin Red solution for5min and then washed with PBS followed by increasing concentrationsof ethanol (70%, 80%, 90%, 100%). For von Kossa stain, cultures werestained with 5% silver nitrate solution under a strong light source forone hour. Cultures were washed three times with water and fixed with5% sodium thiosulfate for 2min.

2.5. Detection of calcium

To quantify calcium content, cells were lysed with radio-im-munoprecipitation assay (RIPA) buffer (1% NP40, 0.5% sodium deox-ycholate, 0.1% sodium dodecyl sulfate, in PBS). The cell lysate wasassayed with Arsenazo III (Genzyme) and the change in absorbancemeasured at 655 nm in an iMark microplate reader (BioRad).Absorbances were compared to a CaCl2 calcium standard and totalcalcium content normalized to the total protein content determined bya Lowry assay as described (Davis et al., 2011). Each biological re-plicate was tested in 5 technical replicates.

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2.6. Image acquisition and analysis

Differentiating cultures were placed inside the Nikon Biostation CT,a hybrid and automated incubator that contains a phase contrast mi-croscope and maintains the culture at 37 °C with 5% CO2. Phase con-trast images were taken every 12 h for a period of 20 days from 10separate areas within the culture plate. Images were assembled tocreate time-lapse videos (Fig. 1). Time lapse videos were analyzed usingthe Matrix Laboratory (MatLab) program, designed to automaticallysegment black calcified areas from the individual phase contrast imagesfrom the time-lapse video using a manual threshold for pixels with anintensity value of 33 or less as described (Martinez et al., 2018). Pixelscorresponding to grey areas, which arise from three-dimensional cellgrowth, were removed to create segmented images of black-appearingcalcified regions only. Remaining segmented pixels were counted toquantitatively represent the degree of calcification from each image.

Concentration response curves for tobacco toxicants were obtainedby normalizing the calcium pixel counts in images of each concentra-tion from a treated sample with the calcified pixel counts from an un-treated sample represented as percentage. To determine the calcifica-tion rate the amount of calcified pixels from each image at a designatedtime point were subtracted from the amount of calcified pixels at t0 anddivided by the hours of elapsed time.

2.7. Exposure with tobacco extract and constituents

Tobacco extract was made by incubating 10 g of Camel or MarlboroSnus in 100ml of DMEM with 15% FBS overnight. The extract wascentrifuged at 450×g for 10min at room temperature and the super-natant again centrifuged at 13,000×g for 1 h to remove finer tobaccodebris. The pH was adjusted to 7.4 and the extract filter sterilized.Nicotine, N′-Nitrosoanabasine (NAB), (R,S)-N-nitroso anatabine (NAT),4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N′-ni-trosonornicotine (NNN), (Toronto Research Chemicals), componentspreviously identified in tobacco (Benowitz et al., 2009; Hecht et al.,1975), were diluted in DMEM to yield a stock solution of 48mM. Celltreatment started with d0 of differentiation and continued throughout.Medium including chemicals freshly diluted to final concentrations waschanged every other day.

2.8. MTT assay

Osteoblast health in response to tobacco extract and constituentexposure was determined by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphe-nylterazolium bromide (MTT) assay. Briefly, cells were incubated withMTT (120mg/ml) at 37 °C for 3 h. After the supernatant was removed,0.04mol/l HCl in isopropanol was added to each well, and the opticaldensity of the solution was read at 595 nm in an iMark™ microplatereader (Bio-Rad) (zur Nieden et al., 2010; zur Nieden & Baumgartner,2010; Walker et al., 2014).

2.9. Determination of nicotine and tobacco-specific nitrosamine content

Concentrations of nicotine and tobacco specific nitrosamines inCamel Snus tobacco extract were measured commercially by EnthalpyAnalytical (Henrico, VA).

2.10. Statistics

All experiments were run in biological triplicate. Arsenazo III-re-agent based calcium assays were run in biological quintuplicate andvideo bioinformatic assessment was performed from a total of 30 areaswithin three independent culture wells. Values represent means± s.d.Statistical assessment was performed with a t-test when appropriate(http://www.graphpad.com/quickcalcs/ttest1/) or One-Way ANOVAwith Holm-Sidak posthoc test when multiple groups were compared(SigmaPlot). A P-value below 0.05 was considered significant. Half-maximal inhibitory doses of cytotoxicity and differentiation (IC50)were taken from concentration-response curves and embryotoxicityclasses calculated according to (Genschow et al., 2002; Genschow et al.,2000).

3. Results

3.1. Snus tobacco extracts are inhibitory to osteogenic differentiation

In culture, calcification from emerging hESC-derived osteoblastspossesses a distinct appearance when viewed under phase-contrastmicroscopy (Fig. 2A) as was previously found for mouse, rhesus, andmarmoset ESCs (zur Nieden et al., 2003; Dienelt & zur Nieden, 2011;Trettner et al., 2014). This signature appearance is viewed as denseblack clusters that result from the inability of light to pass throughcalcified matrix. These areas are immuno-positive for OCN, a bone-specific protein found in the bone matrix, and are composed of calciumas determined by Alizarin Red S (Fig. 2A) (zur Nieden et al., 2003;Sparks et al., 2018; Dienelt & zur Nieden, 2011; Trettner et al., 2014).These dense black areas could be removed with the calcium chelatorEDTA (Fig. 2B), are not present when osteogenic factors were withheldfrom the media (denoted as non-osteogenic) (Fig. 2C, D), and theirincreased levels correlate with an up-regulation in osteoblast-specificgene expression patterns (Sparks et al., 2018).

We next exploited this calcification of the extracellular matrix, as aunique measure of osteoblast function, to determine the potential de-velopmental toxicity of two types of Snus tobacco extract (STE), fromthe Marlboro and the Camel brand and contrasted it to the cytotoxicity,assessed with MTT assay (zur Nieden et al., 2010; zur Nieden &Baumgartner, 2010; Walker et al., 2014). (Fig. 2D). For both extracts,calcification was significantly inhibited in a concentration-dependentmanner when hESCs were tested. Specifically, in a concentration rangebetween 0.1 and 3% STE, the extracts decreased calcification in-dependently of cytotoxicity. In the highest concentrations tested, theextracts were both cytotoxic and inhibitory to differentiation. Humanfibroblasts, who represent fully differentiated somatic cells in thisassay, were even more sensitive to the extracts and began to die atlower concentrations. When applying a biostatistical model that con-trasts the IC50 values of all endpoints (Genschow et al., 2002;

Fig. 1. Schematic overview of image acquisition and data processing for videobioinformatic measurement of calcification. Cells are induced to undergo os-teogenic differentiation through an overgrowth approach followed by additionof osteogenic factors on day 5. Differentiation is captured by time-lapse imagingevery 12 h for a period of 20 days using the Nikon Biostation CT. Time-lapseimages are preprocessed to minimize noise from images. Calcification fromimages is segmented via manual threshold of pixel value set at 33 using MatLab.Numbers of segmented pixels representing calcified regions are quantified re-presenting the amount of calcification from each time point of the time-lapseimaging.

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Genschow et al., 2000) (Fig. 2E), both Snus extracts classified asstrongly embryotoxic, which predicts them to be detrimental to de-veloping skeletal cells.

3.2. Identification of an early detection time point based on calcificationkinetics

While this assay was seemingly predictive of the developmentaladverse effects of Snus, a major disadvantage of it is its long duration.To determine whether assessment could be made earlier than at day 20,we hypothesized that the black appearance of the calcified matrix inphase contrast images could be exploited to measure hESC differ-entiation yield in a kinetic manner, which would allow us to determinea time point in which calcification rose measurably over the back-ground. To do so, live cultures of osteogenically induced and non-os-teogenically induced hESCs were imaged as time-lapse videos using theNikon Biostation CT to follow their commitment into the osteogeniclineage. Images from all time points in the sequence were then ex-tracted from the video and black-appearing calcified areas were seg-mented (Martinez et al., 2018) from all images (Fig. 3A). The first daythat calcification was significantly increased over non-osteogenicallyinduced cultures, as judged from multiple different sets of experiments,was identified to be day 10 (Fig. 3B, C).

To assess differentiation kinetics even further, we next determinedthe calcification rate of the cultures from the time-lapse video (Martinezet al., 2018), which represents the added amount of calcification be-tween to selected time points. Based on this calcification rate, we coulddetermine that the amount of calcification added to the ECM of the H9cells accelerated as time progressed (Fig. 3D, E).

3.3. Snus tobacco extract adversely affects hESC calcification yield andkinetics

We next aimed to measure calcification from time-lapse videos tostudy the embryotoxicity of Camel Snus tobacco extract, the more toxicof the two Snus tobacco products tested, in more detail.Photomicrographs derived from key time-points of time-lapse videoscreated from osteogenically differentiating hESCs treated with STEmorphologically revealed no overt calcium deposition at 1% STE(Fig. 4A) or higher (Fig. 2D).

The image-based calcification assay was then used to quantify cal-cium deposition in concentrations below 1%. The lowest dose tested(0.001% STE) did not cause any significant changes in the number ofsegmented pixels compared to the solvent control cultures (Fig. 4B).However, at doses higher than that, a concentration-dependent de-crease in calcification was noted that was first significant at d8 of

Fig. 2. Osteogenic differentiation and calcification in human ESCs as endpoint for tobacco extract toxicity. (A) Osteogenic differentiation of human ESCs (H9 line)was confirmed on day 20 after differentiation induction by immunocytochemistry of the bone matrix marker osteocalcin (OCN) and deposited calcium ions visualizedby Alizarin Red S staining. Bar= 20 μm. (B) Calcification was removable with 0.5M EDTA. Bar= 100 μm. (C) Determination of calcium content in d20 cultures withArsenazo III reagent, n=5 ± SD. *P < .05, student's t-test. (D) Concentration-response curves for cytotoxicity and differentiation inhibition (d20); n=5 ± SD.ΔP < .05= lowest concentration significantly below the solvent control in the Arsenazo III reagent-based calcium assay determined by One-Way ANOVA.*P < .05= lowest concentration significantly below the solvent control in the MTT assay on H9 hESCs determined by One-Way ANOVA. §P < .05= lowestconcentration significantly below the solvent control in the MTT assay on hFFs determined by One-Way ANOVA. (E) Summary of half-maximal inhibitory con-centrations from concentration-response curves in (D). DAPI, 4′,6-diamidino-2-phenylindole; hFF, human foreskin fibroblast; hESC, human embryonic stem cell;MTT, mitochondrial dehydrogenase activity assay; STE, Snus tobacco extract; OCN, osteocalcin. (For interpretation of the references to colour in this figure legend,the reader is referred to the web version of this article.)

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culture. Typically, toxicity assays will normalize the effect in any givenendpoint to the solvent control (zur Nieden et al., 2010; zur Nieden &Baumgartner, 2010; Walker et al., 2014; Genschow et al., 2002;Genschow et al., 2000). Normalizing the calcification output of a to-bacco treated sample to the solvent control, the image-based programdetected differences between various tobacco concentrations againsupporting the notion that higher doses of STE decreased calcification(Fig. 4C). In addition, both analyses suggested an early delay in calci-fication (d5–10) coupled with a dose-dependent overall yield at thematuration stage (d20). In line with these findings, the calcificationrates showed significantly slower calcification profiles dependent onSTE concentration (Fig. 4D).

We then exploited this image-based kinetic information on calcifi-cation to set a time point for earlier screening than d20, which is cur-rently the norm (zur Nieden et al., 2010; zur Nieden & Baumgartner,2010; Walker et al., 2014; Kuske et al., 2012). To do this, we decided onday 10, since it produced the first stable and significant difference be-yond a baseline threshold (compare Fig. 3B, C). Based on such a d10analysis, a concentration response curve was generated (Fig. 4E) and ahalf-maximal inhibition of differentiation (ID50) determined to be0.12 ± 0.09% STE. A concentration-response curve generated fromd20 data obtained with the traditional dye- based calcification assayrevealed a similar ID50 value of 0.12 ± 0.05% (Fig. 4F).

3.4. Nicotine is not a major contributor to the osteotoxic effect of Snustobacco extract

Tobacco is a complex mixture of at least 5000 chemicals that aretoxic and carcinogenic (Hecht et al., 1975) and it is still undeterminedwhich of these chemicals lead to the observed changes as presentedabove. The most recognized component found in tobacco smoke is ni-cotine, which is responsible for the addictive properties associated withcigarette use (Rickert et al., 1984; Talhout et al., 2011). Its less re-cognized effects include the lowering of 25- hydroxyvitamin D serumlevels as was described for adult female rats suggesting that nicotinemay cause issues with body storage of vitamin D, a hormone necessary

for bone turnover (Fung et al., 1999). Likewise, nicotine caused a re-duction in bone mineral content (Fung et al., 1999). In the adult, ni-cotine can complicate bone healing and may significantly decreasebone mineral content (Gerdhem & Obrant, 2002). In addition, epide-miological studies showed that post-menopausal women who smokelose more cortical bone than non-smokers (Behnke & Smith, 2013),suggesting a link between tobacco and osteoporosis. Thus, as nicotinehas been proposed to influence bone formation previously, we used ourimage-based calcification assay to determine whether nicotine was thecomponent in tobacco responsible for the observed decrease in calcifi-cation.

The results showed no discernible difference in culture morphologyin any of the concentrations of nicotine tested (Fig. 5A). Segmenting thepixels associated with calcification revealed a seeming increase in cal-cification in all tested concentrations, however these were not statisti-cally significant (Fig. 5B, C). Calculations for the calcification rateconfirmed the nicotine treated samples exhibited the same calcificationkinetics as the untreated sample (Fig. 5D). Concentration-responsecurves based on both image analysis and Arsenazo III-reagent calciummeasurement did not show any significant reduction in calcificationyield (Fig. 5E, F). In conclusion, this data implies that the change in thefunctional output of osteoblasts derived from hESCs observed afterexposure to whole tobacco smoke extract was not due to the nicotinecomponent.

3.5. N′-nitrosonornicotine potentially contributes to the toxic effect of Snustobacco extract

Tobacco specific nitrosamines (TSNAs) are another set of majorcomponents in tobacco (Rühl et al., 1980). They are widely accepted tocontribute to its cancer risk (Hecht, 2014; Xue et al., 2014), but theireffect on the osteogenic lineage is less well understood. To determinewhether TSNAs had a contributory effect to the observed develop-mental osteotoxicity of STE, we next treated differentiating hESCs withvarious concentrations of N′-Nitrosoanabasine (NAB), (R,S)-N-nitrosoanatabine (NAT), 4-(methylnitro-samino)-1-(3-pyridyl)-1-butanone

Fig. 3. Determination of earliest measurable difference in calcification yield as endpoint for toxicity assessment. (A) Detection of calcified areas in images fromosteogenic hESC cultures based on segmentation of pixels with a value of ≤33. (B, C) Calcium content of differentiating hESCs measured at 12 h intervals from livecultures (B) and at specific days of the differentiation period (C). (D, E) Calcification rate based on the amount of calcification added in a given hour. (B-E) n=3biological replicates (10 technical replicates ea) ± SD, *P < .05, One Way ANOVA versus the non-osteogenic condition; all subsequent days also exhibit a P valuesmaller than 0.05. STE, Snus tobacco extract.

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(NNK) and N′-nitrosonornicotine (NNN), and measured the outcome ofexposure with regard to cytotoxicity and osteogenic differentiationyield. Human foreskin fibroblasts were also exposed to determine theeffects of the chemicals on fully differentiated cells. The correspondingdata suggested that neither NAT nor NNK caused any adverse effects inthe tested range. In contrast, NAB similarly reduced calcification andcellular viability of the differentiating osteoblasts with similar half-maximal inhibitory doses (Fig. 6A, B).

However, the cytotoxic effect of NAB did not seem to be specific to

developing osteoblasts only, since the fibroblasts were equally sensi-tive. In contrast, NNN decreased calcification output at concentrationsbelow such that caused cytotoxicity in either cell line, suggesting aspecific effect on osteoblast cell fate. The decrease in calcification oc-curred over a large concentration range, with statistically significantdecreases that were lower than the solvent control starting at 85 pg/mlof NNN. Thereby, only NNN exhibited an inhibitory effect on osteoblastdifferentiation at concentrations actually present in the STE extract(Fig. 6A, B).

Fig. 4. Video bioinformatics-based detection of calcification in hESC cultures treated with Camel Snus tobacco extract (STE). (A) Images show a decrease in theamount of calcification observed at 0.1% STE and cell death at 1% STE. (B) Image-based calcification was measured every 12 h. (C) Normalized image-basedcalcification data. (D) Calcification rate calculated from images of tobacco treated samples. (B-D) show selected concentrations only for ease of readability. (E, F) Fullconcentration-response curves determined for all tested concentrations from 10-day-old cultures using video bioinformatics on segmented pixels and from d20cultures using an Arsenazo III-reagent-based calcium assay. B-E n=3 biological replicates 10 technical replicates ea.; F n=3, 5 technical replicates ea. ± SD.*P < .05, One Way ANOVA versus untreated. Statistics shown only for d10 and d20 for ease of readability. STE, Snus tobacco extract.

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Next, we used image analysis on NNN exposed hESCs to determinethe developmental toxicity of NNN in more detail. Visual inspection ofthe segmentation of calcified areas in images of NNN treated cellssuggested that NNN had a concentration-dependent effect on calcifi-cation as already determined with the Arsenazo III reagent-based cal-cium assay (Fig. 7A). This finding was also confirmed with the quan-titative pixel count analysis (Fig. 7B, C). However, the determination ofthe calcification rate discovered a novel parameter of the defect, which

was that low concentrations of NNN were associated with late miner-alization defects, while higher concentrations exhibited early differ-entiation inhibition (Fig. 7D).

Comparison of the image-based calcification assay on d10 (Fig. 7E)with the Arsenazo III reagent-based method as assayed on d20 (Fig. 7F)confirmed the dose-dependent decline in calcification as a result ofNNN treatment with the image-based analysis being slightly moresensitive as the conventional assay. The half-maximal inhibitory dose

Fig. 5. Video bioinformatics time-lapse images and calcium segmentation to study effects of nicotine on hESC osteogenesis. (A) No detrimental effects of nicotine onculture morphology could be detected in photomicrographs of cultures in the tested range. (B) Time-course calcification of nicotine treated osteogenically differ-entiating hESCs as shown every 12 h. (C) Segmented areas in images of nicotine treated samples normalized to the solvent control. (D) Calcification rates ofrepresentative concentrations determined from images taken at 12 h intervals. (B-D) show selected concentrations only for ease of readability. (E) Concentration-response calcification curve across all tested concentrations generated using image-quantified calcium measurement. (F) Arsenazo III reagent-based endpoint cal-cification curve. B-E n= 3 biological replicates 10 technical replicates ea.; F n= 3, 5 technical replicates ea. ± SD. *P < .05, One Way ANOVA versus untreated.Statistics shown only for d10 and d20 for ease of readability.

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that was determined for NNN with the image analysis was 0.115 μg/ml,which was very close to the level of 0.0618 μg/ml that was found in theSTE extract.

4. Discussion

In this study, we have successfully used image analysis and quan-tification of data performed from a series of time-lapsed images in theform of videos to quantify calcification of osteoblasts derived fromhuman ESCs and to characterize the differences in calcification in re-sponse to a major environmental toxicant. The time-lapsed image-basedcalcification assay that was used in this study analyzes the completecalcification process, d0 to d20, from one live culture. The kineticmanner in which it was used facilitated a reduction in assay time whenscreening the effects of tobacco extracts and its constituents on osteo-genesis due to the identification of the earliest time point at whichsignificant differences could be detected between the treated solventcontrol and a tested concentration of chemical. In addition, the calci-fication rate determined that calcification was slightly delayed uponexposure to Snus tobacco extract.

There are four cellular mechanism for the induction of skeletal de-fects during embryogenesis: environmental toxicants may a) change theactivity of metabolic enzymes, b) induce permanent epigenetic changes(Brown et al., 1990), c) reduce cell numbers (McLeod et al., 1972;Snoeck et al., 1990; Matsui et al., 1989) or 4) alter the identity ofprogenitor cells (Godfrey et al., 1994). Evidence from human studies sofar suggest that skeletal development might be programmed as a con-sequence of the first three of these mechanisms. However, the fact thatcalcification reached a limit early on during the differentiation processand failed to further increase over time as seen with the video bioin-formatic approach may provide first evidence that the fourth me-chanism is also at play, as it points to a defect in differentiation early onin development.

Indeed, cigarette smoking has been suggested to have adverse ef-fects on bone tissue. For example, smoking increased the occurrence ofdeveloping osteopathies, such as osteoporosis (Ayo-Yusuf & Olutola,2014; Iqbal et al., 2013; Brook et al., 2012) and Legg-Calve-PerthesDisease (Daniel et al., 2012) and has been implicated in delayed healingof fractured bones (Sloan et al., 2010; Moghaddam-Alvandi et al.,2013). Based on the results presented here, negative effects of tobaccoon the adult bone are not surprising, given that osteogenesis is a life-long process. Bone matrix is constantly digested by the osteoclasts onlyto be renewed by the osteoblasts, in a process called bone remodeling.During bone remodeling, osteoblasts arise from the differentiation ofmesenchymal cells that reside in the bone marrow, which under in-fluence of tobacco may not secrete adequate amounts of bone matrix orundergo apoptosis prematurely.

Our results also corroborate newer data that is suggesting detri-mental effects of prenatal tobacco exposure on the embryonic andnewborn skeleton. Among other environmental factors it accounts forthe high frequencies of many congenital anomalies in infants(Stillerman et al., 2008), including such that affect the skeleton. Limitedresearch in young adults and immature animals suggests a detrimentaleffect of tobacco on bone during growth (Iwaniec et al., 2000a) andmore recent studies correlate in utero exposure to lowered neonatebone mineral quality (Godfrey et al., 2001), consequently leading toincreased childhood fracture risk (Parviainen et al., 2017). We add hereto the growing body of evidence the designation of Snus tobacco extractas a potential developmental toxicant, specifically in the osteogeniclineage.

Our further studies suggested that nicotine was not the primaryculprit causing this adverse effect. In the light of some prior studies ourresults are surprising. In the adult, for instance, nicotine consumptionfrom tobacco use and cigarette smoking can lead to complications inbone remodeling and healing, as determined from a reduction in bonemineral content (BMC). In a cohort study to determine the effects of

Fig. 6. Cytotoxicity and differentiation inhibitioncaused by TSNAs. (A) Differentiation inhibition wasmeasured by Arsenazo III reagent-based calciumassay on d20 of differentiation and contrasted to thecytotoxicity of the chemical in H9 hESCs and humanforeskin fibroblasts also measured on d20. Valueswere calculated as percentage of solvent control andrepresent n=5 ± SD. ΔP < .05, One-Way ANOVAversus solvent control in the Arsenazo III reagent-based calcium assay. *P < .05, One-Way ANOVAversus solvent control in the MTT assay on H9 hESCs.§P < .05, One-Way ANOVA versus solvent controlin the MTT assay on hFFs. (B) Table denoting half-maximal inhibitory concentrations deduced fromgraphical interpolation of data shown in (A) andlisting of TSNA concentrations determined by HPLCanalysis. hFF, human foreskin fibroblast; hESC,human embryonic stem cell; MTT, mitochondrialdehydrogenase activity assay.

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cigarette smoking on bone mass, Gerdhem and Obrant found a sig-nificant decrease in BMC in the femoral neck, tibia, calcaneus, as well asin the lumbar spine and bones in the hand (Gerdhem & Obrant, 2002).Also, in rat calvarial osteogenic cells and clonal mouse calvarial pre-osteoblastic MC3T3-E1 cells, nicotine caused a reduction in ECM cal-cium content (Yuhara et al., 1999). However, the mentioned studiesfound the inhibitory effect of nicotine at relatively high concentrationsof 250 μg/ml. In arterial blood, this concentration is not reached aftersmoking a cigarette (Gourlay & Benowitz, 1997). Concentrations can

reach up to 0.1 μg/ml, but average between 0.02 and 0.06 μg/ml(Gourlay & Benowitz, 1997; Henningfield & Keenan, 1993; Lunell et al.,2000; Rose et al., 1999). While nicotine crosses the placental barrierand is thought to accumulate in fetal serum and amniotic fluid inslightly higher concentrations than in maternal serum (Dempsey &Benowitz, 2001), the in vitro concentrations that cause osteoblasttoxicity are not reached in vivo. In contrast, our study tested nicotine inthese physiological ranges and did not detect any adverse effects.

In line with our findings, other prior literature has also doubted that

Fig. 7. Video bioinformatic based detection of calcification in hESC cultures treated with N′-nitrosonornicotine (NNN). (A) Images show a decrease in the amount ofcalcification with higher concentration of NNN. (B) Image-based calcification was measured every 12 h. (C) Normalized image-based calcification data. (D)Calcification rate calculated from images of NNN treated samples. (B-D) show selected concentrations only for ease of readability. (E) Concentration-response curvedetermined from 10-day-old cultures using video bioinformatics on segmented pixels, n=3 biological replicates (10 technical replicates ea) ± SD. *P < .05, OneWay ANOVA versus untreated. (F) Concentration-response curve from d20 cultures using an Arsenazo III reagent-based calcium assay, n= 3 ± SD. *P < .05, OneWay ANOVA versus untreated. Statistics shown only for d10 and d20 for ease of readability.

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the reproductive toxicity of cigarette smoking is primarily related tonicotine (Dempsey & Benowitz, 2001). Instead, they conclude that thecumulative abnormalities produced by the various toxins in cigarettesmoke are probably responsible for the numerous adverse reproductiveoutcomes associated with smoking. Indeed, even with a sufficientlyhigh intake of nicotine (Akhter et al., 2003), its effects on bone bio-mechanical properties are marginal (Silcox III et al., 1995; Fung et al.,1998; Iwaniec et al., 2000b; Iwaniec et al., 2001), while the completechemical mixture found in tobacco smoke jeopardized bone integrity atan implant interface (Cesar-Neto et al., 2003).

Instead, we show here that during developmental bone toxicity, amajor culprit might be the TSNAs, specifically NNN. While the use ofchewing tobacco reduces exposure to chemicals that are producedduring combustion, Snus still contains a high amount of TSNAs (Songet al., 2016). TSNAs are classified as carcinogens but have also beenshown to cause adverse health effects in mammalian cells due to non-cancerogenic mechanisms, such as promotion of oxidative stress andinflammation (Hoffmann & Adams, 1981; Yalcin & de la Monte, 2016).In fact, efforts are under way to limit TSNA content in Snus and tobaccocompanies are already marketing low TSNA Snus (Song et al., 2016).Designated as such a low TSNA Snus, the extract of the Camel OriginalSnus tested here still contained a level of TSNAs, specifically NNN, thatwas near the half-maximal inhibitory dose found for this chemical.Moreover, NNN was found to have detrimental effects to osteoblastdifferentiation at 85 pg/ml, a concentration much lower than the de-termined half-maximal inhibitory dose and more importantly withinproximity to the range of 8.9–47.2 pg/ml NNN measured in urine oftobacco consumers (Stepanov & Hecht, 2005; Kavvadias et al., 2009).Hence, there is reason for concern that Snus consumption may bedetrimental to differentiating osteoblasts also in the developing embryoand that NNN may be a major contributor to the detrimental effect ofSnus on differentiating osteoblasts.

We conclude this because NNN exposure not only reduced overallcalcification yield, as did Snus exposure, but also because the kineticswith which calcification was affected were very similar. We were ableto deduce the differentiation kinetics due to the time-dependent as-sessment of calcification from video-derived images. Ultimately, thismethod can be used to generate calcification curves and identify ID50 ofother constituents of Snus with a simple variation in the protocol.

Acknowledgements

Experiments in this study were supported by a New InvestigatorAward and a High Impact Pilot Award from the California TobaccoRelated Disease Research Program (TRDRP) [grant numbers 19KT-0017and 25IP-0018] and a grant from the Center for Alternatives to AnimalTesting [grant number 2013-11], respectively. IKCM is a TRDRPDissertation Awardee [20DT-0038] and National ScienceFoundationIGERT fellow of Video Bioinformatics [NSF grant 903667].NRLS acknowledges the support from a TRDRP Cornelius HopperDiversity Award and a fellowship from the International Foundation forEthical Research. BB acknowledges the support from NSF IGERT onVideo Bioinformatics [NSF grant 903667]. JVM is a California Institutefor Regenerative Medicine (CIRM) Bridges fellow. NzN is funded by anRO1 (DE025330) from the National Institutes of Dental andCraniofacial Research. The authors would like to thank the Stem CellCore Facility of the University of California Riverside, a CIRM fundedshared facility, for providing access to the Biostation CT.

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