Psychostimulants Modafinil, Atomoxetine and Guanfacine Impair Bone Cell Differentiation and MSC Migration

Abstract

Attention deficit hyperactivity disorder (ADHD) is one of the most common worldwide mental disorders in children, young and adults. If left untreated, the disorder can continue into adulthood. The abuse of ADHD-related drugs to improve mental performance for studying, working and everyday life is also rising. The potentially high number of subjects with controlled or uncontrolled use of such substances increases the impact of possible side effects. It has been shown before that the early ADHD drug methylphenidate influences bone metabolism negatively. This study focused on the influence of three more recent cognitive enhancers, modafinil, atomoxetine and guanfacine, on the differentiation of mesenchymal stem cells to osteoblasts and on their cell functions, including migration. Human mesenchymal stem cells (hMSCs) were incubated with a therapeutic plasma dosage of modafinil, atomoxetine and guanfacine. Gene expression analyses revealed a high beta-2 adrenoreceptor expression in hMSC, suggesting it as a possible pathway to stimulate action. In bone formation assays, all three cognitive enhancers caused a significant decrease in the mineralized matrix and an early slight reduction of cell viability without triggering apoptosis or necrosis. While there was no effect of the three substances on early differentiation, they showed differing effects on the expression of osterix (OSX), receptor activator of NF-κB ligand (RANKL) and osteoprotegerin (OPG) in the later stages of osteoblast development, suggesting alternative modes of action. All three substances significantly inhibited hMSC migration. This effect could be rescued by a selective beta-blocker (Imperial Chemical Industries ICI-118,551) in modafinil and atomoxetine, suggesting mediation via beta-2 receptor stimulation. In conclusion, modafinil, atomoxetine and guanfacine negatively influence hMSC differentiation to bone-forming osteoblasts and cell migration through different intracellular pathways.

Keywords: ADHD; apoptosis; bone defect; cell migration; hMSCs; osteogenic differentiation.

Figure 1

Transcript expression of human alpha1A-, alpha1B-, alpha1D-, alpha2A-, alpha2B-, alpha2C, beta1-, beta2-, beta3-, dopamine D1, D2, D4-receptors and dopamine- and noradrenaline transporters in hMSC (A) and hOB (B). The gene expressions were each normalized to ha1A-AR (relative gene expression) of hMSCs/hOBs. Shown are means ± standard deviation of three independent experiments with six biological replicates. Significance in difference between alpha-, beta-, dopamine receptors and dopamine- and noradrenaline transporter were determined by ANOVA followed by Tukey’s post hoc tests. *** corresponds to a value of p < 0.001.

Figure 2

Alizarin red staining of undifferentiated hMSC (A) differentiated hMSC (B), modafinil (11.2 µg/mL) treated hMSC (C), atomoxetine (0.9 µg/mL) treated hMSC (D) and guanfacine (17.7 ng/mL) treated hMSC (E) for 28 days in 24-well plates of three independent experiments with six biological replicates. Osteogenic differentiation of hMSC significantly enhanced Alizarin red staining. Modafinil-, atomoxetine- and guanfacine-treated hMSC show a highly significant lower alizarin red staining after 28 days of osteogenic induction compared to differentiated hMSC. Densitometric quantification of Alizarin red staining in hMSC calculated by ImageJ software (F). Significance in difference between undifferentiated, differentiated, modafinil-, atomoxetine- and guanfacine treated hMSC was determined by ANOVA followed by Tukey’s post hoc tests. Scale bar 75 µm. *** corresponds to a value of p < 0.001.

Figure 3

Detection of apoptosis/necrosis in hMSC after administration of modafinil (11.2 µg/mL) (A,B), atomoxetine (0.9 µg/mL) (C,D) and guanfacine (17.7 ng/mL) (E,F) for 24 h, 48 h and 72 h. Briefly, 10% DMSO was added to hMSC as a positive indicator of apoptosis/necrosis. The line diagram represents the luminescent signal of phosphatidylserine (PS) exposure and the fluorescent signal of secondary necrosis in hMSCs. Values are given as mean ± standard deviation to blank of three independent experiments with six biological replicates. Significance in difference between untreated, 10% DMSO-, modafinil-, atomoxetine- and guanfacine treated hMSC was determined by ANOVA followed by Tukey’s post hoc tests. * corresponds to a value of p < 0.05. *** corresponds to a value of p < 0.001.

Figure 4

Quantification of cell viability of hMSC after administration of modafinil (11.2 µg/mL) for 24 h (A), 48 h (B), 72 h (C), atomoxetine (0.9 µg/mL) for 24 h (D), 48 h (E), 72 h (F) and guanfacine (17.7 ng/mL) for 24 h (G), 48 h (H), 72 h (I). Briefly, 10% DMSO was added to hMSC as a positive indicator of cell toxicity. Column height represents the metabolic capacity of hMSC to reduce resazurin into resorufin, which is fluorescent. Values are given as mean ± standard deviation to blank of three independent experiments with six biological replicates. Significance in difference between untreated, 10% DMSO-, modafinil-, atomoxetine- and guanfacine treated hMSC was determined by ANOVA followed by Tukey’s post hoc tests. * corresponds to a value of p < 0.05. *** corresponds to a value of p < 0.001.

Figure 5

Expression of RANKL (A), OSX (B) and OPG (C) transcripts was determined in undifferentiated and osteogenic differentiated hMSC after the administration of modafinil (11.2 µg/mL), atomoxetine (0.9 µg/mL) and guanfacine (17.7 ng/mL) for 28 days by real-time quantitative PCR. Shown are means ± standard deviation of three independent experiments with six biological replicates. Significance in difference between the groups was determined by ANOVA followed by Tukey’s post hoc tests. *** corresponds to a value of p < 0.001. ns: not significant.

Figure 6

Expression of RANKL (A), OSX (B), OPG (C), NANOG (D), OCT-4 (E), SOX2 (F), FABP4 (G), LPL (H), ADIPOQ (I), Aggrecan (J), COL2A1 (K) and SOX9 (L) transcripts were determined in undifferentiated and osteogenic differentiated hMSC after the administration of modafinil (11.2 µg/mL), atomoxetine (0.9 µg/mL) and guanfacine (17.7 ng/mL) for 7 days by real-time quantitative PCR. Shown are means ± standard deviation of three independent experiments with six biological replicates. Significance in difference between the groups was determined by ANOVA followed by Tukey’s post hoc tests.

Figure 7

XCELLigence analysis of real-time cell migration of untreated hMSC and after the administration of modafinil (11.2 µg/mL), atomoxetine (0.9 µg/mL) and guanfacine (17.7 ng/mL) to hMSC for 24 h. XCELLigence measurement of real-time cell migration was performed after the administration of 10 µM ICI-118,551 to modafinil (11.2 µg/mL)-, atomoxetine (0.9 µg/mL)- and guanfacine (17.7 ng/mL) treated hMSC (A–C). Shown are means of normalized cell index ± SD of three independent experiments with six biological replicates.