**1. Introduction**

The last few decades have seen extensive growth in the consumption of dietary supplements (DTs), which can be defined as oral products administered with the purpose of correcting one's dietary deficiencies. DTs containing micronutrients (e.g., vitamins and minerals) play a vital role in satisfying maternal nutritional requirements during pregnancy, which are insufficiently met through their daily diets [1]. Furthermore, micronutrient deficiency during pregnancy has been correlated with serious maternal and fetal health issues, such as congenital malformations and pre-eclampsia [1,2]. Thus, in order to prevent

**Citation:** Rednic, R.; Marcovici, I.; Dragoi, R.; Pinzaru, I.; Dehelean, C.A.; Tomescu, M.; Arnautu, D.A.; Craina, M.; Gluhovschi, A.; Valcovici, M.; et al. In Vitro Toxicological Profile of Labetalol-Folic Acid/Folate Co-Administration in H9c2(2-1) and HepaRG Cells. *Medicina* **2022**, *58*, 784. https://doi.org/10.3390/ medicina58060784

Academic Editor: Masafumi Koshiyama

Received: 6 May 2022 Accepted: 7 June 2022 Published: 10 June 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

possible nutrient inadequacies during pregnancy, DTs are highly recommended by clinicians, becoming a common practice among pregnant women worldwide [1,3]. In particular, folic acid (FA) supplementation (at intake levels of 400 μg to 5 mg/day) is recommended for all women of reproductive age both in the periconceptional period and up until the 12th week of pregnancy [4,5].

FA is a synthetic dietary supplement belonging to a family of water-soluble vitamins typically referred to as "folates" or "vitamin B9" [6,7]. FA plays an essential role in DNA synthesis, repair, and methylation [8], and its maternal supplementation has been correlated with a reduced risk of developing neural tube birth defects [5]. However, FA needs to undergo several transformations within the human body in order to become metabolically active [9]. This process includes the reduction of FA to dihydrofolate (DHF) and tetrahydrofolate (THF), followed by its conversion to the biologically active 5-methyltetrahydrofolate (5-MTHF) [6]. 5-MTHF represents the predominant form found in plasma (>90% of total folate) and the main active metabolite of the ingested FA [9].

Many physiological changes occur during pregnancy to enable proper placental and fetal development. Unfortunately, these changes might affect preexisting maternal diseases or even result in pregnancy-related disorders [10]. Hypertension represents the most commonly encountered medical complication during pregnancy (up to 10% of pregnancies) and is the leading cause of maternal, fetal, and neonatal morbidity and mortality worldwide [11–13]. Pregnancy-related hypertensive disorders, which have been associated with an increased risk of developing maternal type 2 diabetes and cardiovascular disease in later life [14,15], cover a broad spectrum of conditions, including chronic hypertension, gestational hypertension, pre-eclampsia, and pre-eclampsia superimposed on chronic hypertension [16,17]. While the definitive treatment for acute hypertensive syndromes occurring during pregnancy is delivery, antihypertensive medication is one of the most employed management strategies in preventing maternal cerebrovascular and cardiac complications. Antihypertensive agents that are widely recommended to control maternal hypertension during pregnancy should not impair the uteroplacental and fetal circulation, and present limited toxicity to the fetus [16]. In current practice, the first-line pharmacological treatment of pregnancy-related hypertensive disorders is based on antihypertensive drugs, such as methyldopa and labetalol, while the second-line strategy includes nifedipine, verapamil, clonidine, and hydrochlorothiazide [18].

Labetalol (LB) is a β-blocker medication commonly recommended as a safe option for the treatment of maternal hypertension during pregnancy [19,20]. Compared to other βblockers, LB contains both selective α-adrenergic and non-selective β-adrenergic blocking activities in a single agent and preserves the uteroplacental blood flow. However, despite its favorable safety profile, LB has been associated with several side effects, including hypotension, bradycardia, cardiac impairment, and maternal hepatotoxicity [21,22].

The leading hypothesis of this study was that labetalol-folic acid or folate co-administration during pregnancy might result in deleterious side effects, which, to the best of our knowledge, lack any investigation so far. Thereafter, the aim of the current paper was to portray an in vitro toxicological profile of labetalol associated with folic acid and folate using healthy myoblasts and hepatocytes as models for compound-induced cardio- and hepato-toxicity.

### **2. Materials and Methods**

### *2.1. Reagents*

Labetalol hydrochloride (LB), folic acid, folate (as 5-Methyltetrahydrofolic acid), trypsin-EDTA solution, phosphate saline buffer (PBS), dimethyl sulfoxide (DMSO), fetal calf serum (FCS), penicillin/streptomycin, insulin from bovine pancreas, hydrocortisone 21-hemisuccinate sodium salt, and MTT (3-(4,5 dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide) reagent were purchased from Sigma Aldrich, Merck KgaA (Darmstadt, Germany). Dulbecco's Modified Eagle Medium (DMEM; ATCC® 30-2002™) was purchased from ATCC (American Type Cell Collection, Lomianki, Poland), and William's E Medium was purchased from Gibco Waltham, MA, USA.

### *2.2. Cell Culture*

Myoblast (heart, myocardium) immortalized cell line (H9C2(2-1); code CRL-1446™) was provided as a frozen vial by ATCC. The cells were cultured in their specific media (DMEM). Hepatic immortalized cell line (HepaRG; code HPRGC10) was purchased from ThermoFisher Scientific (Gibco Waltham, MA, USA) and cultured in William's E Medium enriched with insulin and hydrocortisone 21-hemisuccinate sodium salt at final concentrations of 4 μg/mL and 50 μM, respectively. Both media contained a 10% FCS and 1% penicillin (100 U/mL)/streptomycin (100 μg/mL) mixture. The cells were kept in an incubator at 37 ◦C and 5% CO2 during the experiments.

### *2.3. Cellular Viability*

To evaluate the impact of LB, FA, and FT on the viability of myoblasts and hepatocytes, the MTT technique was applied. Briefly, H9c2(2-1) and HepaRG cells were cultured in 96-well plates (104 cells/well) and stimulated for 72 h with LB (10, 25, 50, 100, and 150 nM), FA, and FT (0.2, 1, 10, 25, and 50 nM). The stock solution of LB was prepared by dissolving the substance in distilled water, while the FA and FT stock solutions were prepared in DMSO. The FA and FT solutions were further diluted in distilled water and culture media for in vitro testing. At the end of the treatment, fresh media (100 μL) and MTT reagent (10 μL) were added to the wells, and the plates were incubated at 37 ◦C for another 3 h. Finally, the solubilization solution (100 μL/well) was added to each well. The plates were kept at room temperature for 30 min, protected from light, and the absorbance values were measured at two wavelengths (570 and 630 nm) using Cytation 5 (BioTek Instruments Inc., Winooski, VT, USA).

### *2.4. Cellular Morphology and Confluence*

To verify the influence of LB-FA and LB-FT associations on the morphology and confluence of H9c2(2-1) and HepaRG cells, a bright field microscopic examination was performed. The cells were photographed at the end of the 72-h treatment period using Cytation 1 (BioTek Instruments Inc., Winooski, VT, USA). The obtained pictures were processed using the Gen5™ Microplate Data Collection and Analysis Software (BioTek Instruments Inc., Winooski, VT, USA).

### *2.5. Wound Regeneration*

The regenerating potential of LB, FA, FT, and their combinations on healthy H9c2(2-1) and HepaRG cells following wounding was evaluated by applying the wound healing (scratch) assay. In brief, the cells (105 cells/mL/well) were cultured in 12-well plates, and a manual scratch was made in the middle of each well. The cells were treated with the test compounds for 24 h, and representative images were taken at 0 h and 24 h using an Olympus IX73 inverted microscope equipped with a DP74 camera. The wound widths were measured at the end of the treatment with CellSense Dimension 1.17 (Olympus, Tokyo, Japan). The quantification of the effects in terms of cell migration was performed by calculating the wound healing rates (%) according to a formula used in our previous work [23,24].

### *2.6. Statistical Analysis*

All data are expressed as the means ± SD, and the differences were compared by one-way ANOVA analysis followed by Dunnett's multiple comparisons post-test. The used software was GraphPad Prism version 9.2.0 for Windows (GraphPad Software, San Diego, CA, USA, www.graphpad.com). The statistically significant differences among the data are marked with \* (\* *p* < 0.05; \*\* *p* < 0.01; \*\*\* *p* < 0.001; \*\*\*\* *p* < 0.0001).
