Plant-Derived Diamine Oxidase Modulation of Histamine-Induced Ca²⁺ Release in Intestinal Caco-2 Cells: A Cellular System to Evaluate Its Histaminase Efficacy

Abstract

Highlights

• Histamine induces Ca²⁺ release in enterocyte-like Caco-2 cells via H1 receptors (H1R).

• Histamine Ca²⁺ stimulation is fast (less than 50 s), cumulative and repetitive (after cell washings).

• Diamine oxidase (DAO) disruption of histamine reduces H1R–mediated Ca²⁺ signaling.

• Vegetal (vDAO) exerts a comparable inhibition of Ca²⁺ release as desloratadine, but at a lower concentration.

• Oral forms of vDAO may alleviate some intestinal histamine-related dysfunctions

Abstract

Histamine accumulation is associated with allergic reactions and intestinal dysfunction through activation of H1R Receptors (H1Rs) and subsequent intracellular Ca²⁺ mobilization. Although antihistamines such as desloratadine block H1Rs to alleviate symptoms, they may induce adverse effects (dizziness, nausea, neurological disorders). Diamine oxidases (DAOs) catalyze the oxidative deamination of histamine to imidazole acetaldehyde with consumption of oxygen and concomitant production of hydrogen peroxide and ammonia. Instead of blocking H1R, we have proposed vegetal diamine oxidase (vDAO) as an enzymatic approach to decompose histamine. This study investigated whether vDAO, by degrading histamine, could modulate histamine-induced Ca²⁺ mobilization compared with desloratadine. Using intestinal Caco-2 cultured cells loaded with the fluorescent Ca²⁺ indicator Fluo-4 AM, intracellular calcium (Ca²⁺) dynamics were analyzed by confocal microscopy following histamine stimulation. vDAO significantly downregulated histamine-induced Ca²⁺ mobilization. Notably, at a fixed histamine concentration sufficient to trigger Ca²⁺ release, vDAO achieved the same level of calcium mobility inhibition at a concentration three-fold lower than that of desloratadine, suggesting a high efficiency of DAO. These findings indicate that histamine degradation with orally administrable DAO may reduce Ca²⁺ signaling in intestinal epithelial cells, providing a promising approach to reduce histamine-related damage or a reinforcement of current antihistamine anti-inflammatory medication based on histamine receptor blockade.

1. Introduction

Histamine is a biogenic amine, endogenously generated by decarboxylation of his-tidine by histidine decarboxylase in various eukaryotic cells (i.e., mastocytes, basophils, gastric enterochromaffin-like cells, neurons) and in some bacteria of the intestinal microbiota [1,2,3,4]. Histamine is also exogenously provided by the intake of fermented food and beverage [5,6,7]. This aromatic amine is involved in many biological processes including the control of gastrointestinal secretion [8], inflammation, allergic and pseudo-allergic reactions [9,10], neurotransmission [11], intestinal cell proliferation, migration and differentiation [12,13] and gut contractibility [14]. Specifically, higher levels of histamine have been associated with gastrointestinal (GI) dysfunctions (Crohn’s disease, ulcerative colitis, irritable bowel syndrome, histamine intolerance), neurological impairment (cognition, sleep–wake cycle, nociception), and tumor development and progression [8,15,16,17,18]. The effects of histamine are mediated by histamine receptors H1 to H4 which activate cellular G protein signaling pathways [19,20]. H1Rs, of interest in this study, are expressed by a broad range of mammalian cell types including enterocytes [21,22].

The enterocyte-like Caco-2 cells isolated from human colon carcinoma are frequently used as an in vitro model mimicking human gut properties [23,24] in investigation of intestinal epithelium features.

Diamine oxidase (DAO), also known as histaminase (EC 1.4.3.6), is an oxidoreductase that catalyzes the oxidative deamination of primary amino groups in certain biogenic amines such as histamine, tyramine, putrescine, and cadaverine [25,26,27] yielding the corresponding aldehydes along with production of hydrogen peroxide and ammonia (Scheme 1).

Scheme 1.

Oxidative decomposition of histamine catalyzed by DAO and disruption of H₂O₂ catalyzed by Catalase.

In the intestinal lumen, an endogenous DAO is released from enterocytes [14,28] and may be reinforced or completed with orally administered food supplements as tablet forms of DAO from pig kidney [29,30,31] or from plants such as Lathyrus sativus or Pisum sativum [32,33]. The endogenous intestinal DAO may be inhibited in certain individuals due to an intestinal dysbiosis that could damage the intestinal mucosa where DAO is produced [34]. A low activity of intestinal DAO may also be due to genetic factors such as nucleotide polymorphisms of the AOC1 gene that encodes the DAO enzyme [35]. Furthermore, several types of drugs such as chloroquine, clavulanic acid, cimetidine, verapamil are known to generate 50% (or more) inhibition of DAO [36]. Also, metformin, the most prescribed and consumed oral anti-diabetic drug, inhibits DAO in the intestinal tract [37]. In rare conditions, food histamine may generate scombroid poisoning, anaphylaxis and even death [38]. This histamine intoxication is not an allergy but rather a so-called “pseudo allergy”, a histamine intolerance (HIT) often due to diamine oxidase (DAO) deficiency [27]. For all these conditions and medications, orally administered plant-derived DAO may be beneficial to control and prevent histamine-related dysfunctions.

Histamine dysfunctions may be related to an inhibition of endogenous DAO or to common intoxication with histamine from food (frequently fermented food such as sauerkraut or pickles, or food with high histamine levels such as some wines or cheeses). This may trigger symptoms similar to allergic phenomena (pseudo-allergies) or even major allergic conditions, including anaphylaxis [39,40]. Unbalanced histamine levels are quite frequent in food poisoning in allergic phenomena or in cases of histamine-related diseases (such as mastocytosis), which emphasizes the importance of administration of antihistamine drugs or/and of DAO supplements (particularly for intestinal dysfunctions). In recent decades, the intake of oral forms (capsules, tablets) containing DAO purified from pig kidney has been reported to mitigate symptoms in patients affected by food histamine intolerance [12,27,41,42].

Vegetal DAO (vDAO), with an activity several times higher than that from pig kidney, was proposed as an additional tool to alleviate intestinal histamine-related dysfunctions [43]. The efficacy of vDAO to countect the histamine effect was showed in various in vitro cellular systems [13,14,27].

This report aims to investigate the efficiency of DAO to inhibit the histamine-triggered intracellular Ca²⁺ mobility and to compare its efficiency to that of the currently used antihistamine drug desloratadine (Des).

We have previously reported the capacity of vDAO to decrease the histamine-triggered colon contractions on an ex vivo murine model [14] and we have now investigated mechanistic aspects of vDAO controlling the histamine-induced Ca²⁺ mobility on intestinal Caco-2 cells, in comparison with the antihistamine drug Des. The control of Ca²⁺ mobility in epithelial enterocytes is important because they can modulate the release of calcium into the circulation and also from the epithelium to the lumen. Since Ca²⁺ mobility is enhanced by high histamine level, it was of interest to investigate the capacity of DAO (Histaminase) to limit the histamine-induced Ca²⁺ mobility and related intestinal motility that can be painful, particularly in inflammatory disorders.

2. Materials and Methods

2.1. Materials

Aminoantipyrine (AAP); bovine serum albumin (BSA); calcium chloride (CaCl₂); ethylenediaminetetraacetic acid (EDTA) disodium salt; sodium 3,5-dichloro-2-hydroxybenzenesulfonate (DCHBS); 1,4-diaminobutane dihydrochloride (putrescine); dibasic sodium phosphate salt (Na₂HPO₄) dihydrate; glutamine; 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid (HEPES); histamine; horseradish peroxidase (HRP) type I; monobasic sodium phosphate salt (NaH₂PO₄) dihydrate; and probenecid were purchased from MilliporeSigma (Oakville, ON, Canada). Fluo-4 acetoxymethylester (Fluo-4 AM) and pluronic acid were purchased from Thermo Fisher Scientific (Mainway, Burlington, ON, Canada). Dulbecco’s modified eagle medium (DMEM) containing 25 mM glucose, 1× non-essential amino acids and 10,000 U/mL penicillin–streptomycin were purchased from Hyclone (Logan, UT, USA); fetal bovine serum (FBS) was from Wisent Bioproducts (St-Bruno, QC, Canada). The colorectal adenocarcinoma (Caco-2) cell line was sourced from Dr. A Zweibaum [44]. All current chemicals were reagent grade and were used without further purification.

2.2. Vegetal Diamine Oxidase Extraction, Purification and Characterization

Diamine oxidase (DAO) was purified from etiolated Lathyrus sativus seedlings as reported by [14], previously stored at −80 °C. All procedures were performed at 4 °C to preserve enzyme activity. Frozen seedlings (around 500 g fresh weight) were gently stirred in an equal volume (w/v) of extraction buffer consisting of 50 mM sodium phosphate buffer (pH 5.5) and 0.2 M NaCl and then filtered through cheesecloth and the filtrate was centrifuged at 15,000× g for 30 min at 4 °C. The supernatant (around 200 mL) was collected as the crude enzyme extract that was loaded onto an ion exchange column (YMC-BioPro S75) previously equilibrated with 50 mM sodium phosphate buffer (pH 5.5). Chromatography was performed at a flow rate of 1 mL/min. Fractions (25 mL) were collected and monitored by absorbance at 280 nm. Fractions exhibiting diamine oxidase activity were identified by hydrogen peroxide production during putrescine oxidation using a Lambda 750 spectrophotometer (Perkin Elmer, Shelton, CT, USA). DAO activity was assayed at 25 °C in 0.1 M sodium phosphate buffer with pH 7.2 by following the formation of a pink product (ε_515nm = 26,000 M⁻¹cm⁻¹) generated from the oxidation of 1 mM DCHBS by hydrogen peroxide (H₂O₂), produced from the oxidation of 3 mM putrescine, in the presence of 10 μg/mL HRP, and subsequent condensation with 0.1 mM AAP [13,45]. One enzyme unit (U) oxidizes one μmole putrescine/min.

Enzyme-containing fractions were pooled and concentrated by ultrafiltration using centrifugal filter units (MerckMillipore, Kirkland, QC, Canada) with a weight cut-off of 10–30 kDa. The purity of the DAO preparation was evaluated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions [43,45,46,47], DAO activity was measured spectrophotometrically [48], and protein concentration was determined by the Bradford colorimetric method at 595 nm [49] using bovine serum albumin (BSA) as a standard. Following purification, the enzyme preparation was lyophilized. The resulting purified preparation yield was 305 ± 13 mg of dry powder, corresponding to a protein content of 0.38 ± 0.08 mg protein per mg solid and a specific activity of 23.8 ± 3.3 U/mg solid. The lyophilized enzyme was stored at −80 °C.

2.3. Caco-2 Cell Culture

Caco-2 cells (passages 207 to 255) were cultured in DMEM supplemented with 25 mM glucose, 15% heat-inactivated FBS (56 °C, 30 min), 0.1 mM non-essential amino acids, and 50 U/mL penicillin—50 μg/mL streptomycin. Cells were maintained in 75 cm² culture flasks at 37 °C in a humidified atmosphere containing 5% CO₂. The passage of cells was performed weekly using 0.05% trypsin supplemented with 0.053 mM EDTA, and the culture medium was renewed every two days. For experimental procedures, cells were seeded in 6-well plates at a density of 2 × 10³ cells/well and cultured for 21 days to allow differentiation [24,50].

2.4. Calcium (Ca²⁺) Flow Measurements

Differentiated Caco-2 cells (21 days) were incubated at 37 °C for 1 h with 1 µM non-fluorescent Fluo-4 acetoxymethyl ester (Fluo-4 AM) calcium (Ca²⁺) indicator, 1µM Pluronic acid and 2.5 mM probenecid, all prepared in 10 mM HEPES buffer (pH 7.2). After incubation, cells were washed three times with HEPES buffer to remove excess extracellular fluorophore. Cells were then incubated with 100 μL of 10 mM HEPES buffer for an additional 30 min to allow intracellular esterases to convert Fluo-4 AM into its active fluorescent form, Fluo-4.

Fluo-4 AM is a membrane-permeant probe able to diffuse passively across the plasma membrane due to its hydrophobic AM ester groups. After entering cells, cytosolic esterases cleave its AM groups, producing Fluo-4 fluorescent indicator. Upon binding intracellular Ca²⁺, Fluo-4 exhibits increased fluorescence intensity, enabling sensitive detection and quantification of dynamic changes in Ca²⁺ concentrations [51,52].

In the current study, the Caco-2 cells were stimulated with histamine. Fluorescence measurements obtained in the absence of vDAO or of desloratadine (Des) are considered as controls. Thus, the resulting intracellular Ca²⁺ mobilization responses enabled a direct comparison of the in vitro effects of vegetal DAO (histaminase) and of Des (antihistamine) in modulating histamine-induced Ca²⁺ signaling.

Confocal microscopy was used to assess the effect of vDAO purified from Lathyrus sativus seedlings on histamine-induced Ca²⁺ mobilization in 21-day differentiated Caco-2 cells. To track intracellular calcium release, Caco-2 cells loaded with Fluo-4 AM were exposed to histamine (final concentrations in the range of 0.05–2.50 mM) and vDAO (concentrations in the range of 5–17 μM) or Des (concentrations in the range of 5–20 μM).

Imaging was performed in a humidified 5% CO₂–air atmosphere at 37 °C, under a Nikon A1 confocal microscope (Nikon corporation, Melville, NY, USA) equipped with a 10× objective. Fluo-4 Ca²⁺ fluorescence was detected at an excitation wavelength (λ_Exc) of 488 nm and an emission wavelength (λ_Em) of 525 nm, as previously described [51,52]. Images were acquired in kinetic scan mode for up to 2.5 min, with scans every 2 s, using a pinhole diameter of 11.9 µm, a detector high voltage (HV) of 45, and an offset of −5.

In selected experiments, fluorescence was recorded in real time. Quantitative analysis was performed using Fiji (ImageJ 1.54f software, http://imagej.org, accessed: 10 February 2025). Regions of interest (ROIs) were defined to quantify intracellular fluorescence, and data were expressed as area under the curve (AUC) from time-resolved recordings. Results are presented as mean ± SEM from at least 3 independent experiments. To evaluate inhibitory effects, various concentrations of DAO or Des were co-administered with a fixed concentration of histamine. A washing step was also performed to evaluate the reproducibility of the histamine-induced response.

To better understand the aspects related to histamine triggering Ca²⁺ mobility and the inhibition provided by DAO, the effect of H₂O₂ alone on Ca²⁺ mobility was followed as the control. The amounts of H₂O₂ generated, in three min, at the oxidation of 1.25 mM histamine with increasing concentrations (5–17 μM) vDAO (same concentrations used to evaluate the Ca²⁺ mobility) were determined by UV/Vis spectrophotometry. Separately H₂O₂ alone (no histamine, no vDAO) at same concentrations as those determined at histamine oxidation was added to cultured Caco-2 cells and Ca²⁺ mobility was evaluated with the Fluo-4 AM sonde by confocal microscopy. Enzyme and H₂O₂ solutions were prepared in 10 mM HEPES buffer, with pH 7.2.

2.5. Statistics

Data are expressed as mean ± SD of at least n = 5 different measurements. Statistical tests were performed with the GraphPad Prism 9.5.1, using either one-way ANOVA (for comparison between three groups) or two-tailed Student’s t-test) (for comparison between two groups). Statistical significance was defined as *p < 0.1 and **p < 0.01.

3. Results and Discussion

The 21-day-old differentiated Caco-2 cells represent an accepted model which correlates well with the permeability barrier function of the intestinal epithelium cells [53,54]. Histamine activation of H1 receptors (H1Rs) regulates the Ca²⁺ intracellular concentration and its pathways in most of the intestinal cells [55].

3.1. Histamine-Induced Ca²⁺ Mobility in 21-Day Differentiated Caco-2 Cells

Fluo-4 AM, a sensitive probe, is used as an indicator in Ca²⁺ mobilization experiments [56,57]. In the absence of histamine stimulation, Caco-2 cells, loaded with Fluo-4, showed only a few green fluorescent spots under confocal microscopy (Figure 1A1–A3).

Figure 1.

Ca²⁺ mobilization in 21-day differentiated Caco-2 cells loaded with Fluo-4. (A): Non-stimulated cells; the green fluorescence (A1), transmitted light (A2) and (A1,A2) are merged into (A3). (B): time-course of cells stimulated with 0.625 mM histamine showing a fast (seconds) increase (B1–B4) of green fluorescence (red scale bar = 100 μm). One experiment is representative of 5. (C) Quantitative representation of relative fluorescence (RFU) increases with the time from 0 to 60 s. Mean ± SD, n = 5 independent measurements; * p < 0.1, ** p < 0.01.

The very limited, almost non-fluorescent zone (Figure 1A1) could have been related to the absence of living cells. However, under transmitted light microscopy, a well-defined cellular layer appeared with some cellular clusters (Figure 1A2). Merging Figure 1A1, A2 into A3, with overlap of the two panels (A1 and A2), confirmed that the majority of green spots (Figure 1A1) are associated with clustered cells (Figure 1A2) probably due to the aggregative properties of Caco-2 cells [53]. The loading of Fluo-4 AM was confirmed in the absence of histamine by a basal (very low) fluorescence with only a few fluorescent spots (Figure 1A3). As expected, this basal fluorescence was comparable to that observed at time zero of the cell’s treatment with histamine (Figure 1B1). The cell’s treatment with histamine (0.625 mM) resulted in a time-dependent increase in the number of green fluorescent spots, indicating a fast activation (less than 1 min) of Ca²⁺ flow to the cytosol occurred in the cells at histamine signaling (Figure 1B1–B4).

Fluorescence quantification derived from Figure 1A is shown in Figure 1C: a fast increase in intracellular free Ca²⁺ content was noticed at about 40–50 s (s) and became significant at 60 s following histamine stimulation. The rapid increase in fluorescence followed by a sustained plateau (Figure 1B) is consistent with intracellular Ca²⁺ release from endoplasmic reticulum stores and subsequent stabilization through homeostatic mechanisms.

Sequential applications of histamine 0.625 mM were used to evaluate the reproducibility and kinetics of the fluorescence response. Figure 2A shows the temporal fluorescence profile during consecutive applications (first and second) of 0.625 mM of histamine, while Figure 2B shows quantification of the cumulative fluorescence responses to histamine stimulation (1 and 2). Importantly, Figure 2C,D showed that removal of extracellular histamine by washing leads to a marked decrease in fluorescence toward baseline levels. Subsequent re-stimulation with histamine induces a response of comparable amplitude, indicating that the signal is reversible and not due to cumulative artifacts. Together, these results confirm that the increase in fluorescence is specifically driven by extracellular histamine and reflects a reproducible, receptor-mediated Ca²⁺ response.

Figure 2.

Histamine stimulations: cumulative (A,B) and repetitive with alternated washings (C,D). (A) Time-course of Fluo-4 fluorescence upon 0.625 mM histamine subsequent cumulative treatments (one experiment representative of 5). (B) Quantitative analysis of Fluo-4 fluorescence as cumulative area under the curve (AUC) at 0.625 mM histamine stimulations (mean ± SD, n = 5 independent measurements, p > 0.01). (C) Time-course of Fluo-4 fluorescence upon 0.625 mM histamine subsequent repetitive treatments alternated with washings (PBS 1×, pH 7.2). (D) Quantitative analysis of Fluo-4 fluorescence as area under the curve (AUC) at repeated 0.625 mM histamine stimulation after intermediate washing (mean ± SD, n = 5 independent measurements, p > 0.01).

The concentration of 0.625 mM histamine used on Caco-2 cells to induce Ca²⁺ mo-bility appears higher compared to commonly used concentrations in histamine-induced signaling studies in other experimental models. For instance, in a previous ex vivo study (murine distal colon muscle) on DAO alleviating the histamine-induced intestinal contraction, the histamine concentration to trigger intestinal motility was much lower (50 μM) [14]. In the current in vitro experimental conditions on intestinal Caco-2 cultured cells, such concentrations did not generate a sufficiently robust and reproducible intracellular Ca²⁺ answer for quantitative analysis. Therefore, a higher concentration (0.625 mM) was retained to obtain consistent and measurable responses above baseline noise. This histamine concentration is comparable with that used in a number of other studies on Caco-2 cells that elicit responses to histamine at relatively high concentrations (1 mM) and with a lethal concentration (LC₅₀) of ≅ 10 mM following 24 h of exposure [58]. These results showed that washing steps restore baseline fluorescence. Removal of extracellular histamine resulted in a decrease in fluorescence toward baseline levels. A second histamine application induced a response comparable to the previous one, indicating the signal reversibility.

The fluorescence intensity associated with intracellular Ca²⁺ mobilization increased in a concentration-dependent manner following histamine stimulation (Figure 3A). Nonlinear regression analysis yielded a half effective concentration (EC₅₀) of 0.140 mM (95% confidence interval (CI), 0.09–1.30 mM) (Figure 3B). These results indicate that histamine induces intracellular Ca²⁺ signaling in Caco-2 cells within the high micromolar to low millimolar concentration range. Accordingly, a histamine concentration of 1.25 mM histamine was selected for the _VDAO and Des assays to ensure a robust and reproducible cellular response. Notably, this concentration falls within the upper limit of the EC₅₀ 95% CI, providing additional statistical support for its experimental use.

Figure 3.

Effect of histamine on Ca²⁺ mobility in 21-day differentiated Caco-2 cells loaded with Fluo-4 AM. (A) Kinetics of Fluo-4 fluorescence upon treatment with histamine at different concentrations. For each concentration, one experiment representative of 5 is presented. (B) Effect of histamine concentration on the Fluo-4 fluorescence expressed as AUC after subtraction of control values. (Mean ± SD; n = 5 independent measurements.)

Histamine as a multifactorial bioactive agent, when in excess (from food or gut bacteria) or when intestinal DAO is impaired, may exert deleterious effects locally and via diffusion into the bloodstream. Histamine promotes inflammation by attracting immune cells (mast cells, neutrophils) and may trigger abdominal pain and diarrhea by activating visceral neurons [6,59]. Histamine, as an inflammatory mediator, modulates the increase in epithelial permeability [60] by disrupting the tight junctions between intestinal cells. Thus, histamine will diffuse into the bloodstream causing systemic effects, including pseudo-allergy phenomena. This process can be prevented by orally administering DAO.

3.2. _VDAO and Des Prevented Ca²⁺ Mobility Stimulated by Histamine

An endogenous DAO produced by Caco-2 cells has previously been reported [28,58,61,62]. In the current study, all data were recorded considering control conditions in the absence of exogenous vDAO or of Des. Thus, the observed responses in Ca²⁺ mobility stimulated by histamine were ascribed to vDAO or to treatment of cells with Des. A fixed concentration (1.25 mM) of histamine was selected for the subsequent experiments to evaluate the effect of vDAO on Ca²⁺ mobility. At this concentration, histamine showed a low toxicity (about 78–80% viability on cultured Caco-2 cells). The vDAO was found to generate a concentration-dependent protective inhibition of Ca²⁺ mobility triggered by 1.25 mM histamine (Figure 4A). For instance, vDAO at a final concentration of 17 μM produced about 96% inhibition of Ca²⁺ mobilization (Figure 4B).

Figure 4.

Effect of vDAO and of Des on histamine-induced Ca²⁺ mobilization in 21 days-differentiated Caco-2 cells loaded with Fluo-4. (A) Time-course of Fluo-4 fluorescence at Ca²⁺ mobilization triggered by 1.25 mM histamine, under treatment with vDAO at various concentrations (each experiment is representative of 5); (B) quantitative analysis of Fluo-4 fluorescence decrease at treatment with different concentrations of vDAO, expressed as AUC (mean ± SD, n = 5 independent experiments); (C) time-course of Fluo-4 fluorescence at Ca²⁺ mobilization triggered by 1.25 mM histamine at treatment with desloratadine (Des) at various concentrations (each experiment is representative of 5); (D) quantitative analysis of Fluo-4 fluorescence decrease at treatment with different concentrations of Des, expressed as AUC (mean ± SD, n = 5 independent experiments); (E) effect of vDAO at various concentrations on Fluo-4 fluorescence when cells were not exposed to histamine (one experiment representative of 5); (F) effect of Des at various concentrations on Fluo-4 fluorescence when cells were not exposed to histamine (one experiment representative of 5).

The effect of Des on histamine-induced Ca²⁺ release was also evaluated. At a con-centration of 50 μM Des, the Ca²⁺ flux was 72% inhibited (Figure 4C). These data indicate a stronger inhibitory effect of vDAO compared to Des (Figure 4D). Practically, vDAO presented a higher potency in reduction in Ca²⁺ mobilization, with a Half Maximal Inhibitory Concentration IC₅₀ of 7.8 μM, which was three-fold lower compared to the IC₅₀ of 24.6 μM for Des (Figure 4B,D). This effect is in agreement with the previous observation of Limon et al. [63] showing a moderate inhibition of intracellular Ca²⁺ flux at increasing concentrations of Des [63,64].

Our data showed that in the absence of histamine, neither vDAO nor Des induced fluorescence of Fluo-4 probe in Caco-2 cells (Figure 4E,F). In this case, the lack of effect of vDAO or of Des on Ca²⁺ mobility can be considered as a negative control.

Similar protective effects were previously found with vDAO on histamine-induced intestinal smooth muscle contractibility [14], but with an ex vivo model measuring motility of distal colon segments (triggered by histamine and alleviated by DAO). The current in vitro study on cultured intestinal Caco-2 cells and confocal microscopy measurements shows the histamine-induced Ca²⁺ flux dynamics counteracted by DAO. This in vitro model for investigating histamine effects in intestinal cells is much more convenient and easier to apply than the ex vivo model, particularly for the aspects related to Ca²⁺ mobility.

The stability of DAO is related to its concentration and to the nature of the environmental fluids [48]. In this way it was crucial to eliminate possible interference and to choose the best experimental conditions. The DMEM is a rich cell culture medium supplemented with antibiotics, with non-essential amino acids and with fetal bovine serum (FBS) which is a main component of eukaryote cell culture media. The FBS collected from bovine fetal blood [65] may contain serum amine oxidases (SAOs) as well as DAO, which are known to be highly expressed in the placenta [66]. Consistent with this possibility, vDAO activity in the presence of FBS was greater than that measured in the control 0.1 M phosphate buffer (pH 7.2) (Figure S1), suggesting the use of FBS-free medium to avoid interference in further experiments.

It was also of interest to evaluate the vDAO activity as a function of increasing concentrations of histamine. An increase in the reaction rate was observed with histamine concentrations ranging from 0.01 to 10 mM and a slight inhibition was measured at a high concentration (100 mM) of histamine, probably due to substrate inhibition (Figure S1).

A noteworthy feature of histamine oxidation by vDAO is the concomitant production of hydrogen peroxide (H₂O₂) (Scheme 1). Table S1 provides supplementary data on the effect of vDAO and histamine on production of H₂O₂ and on Ca²⁺ mobility (Table S1A) and on the effect of H₂O₂ alone (as a control) at the same concentrations, but in the absence of vegetal DAO and of histamine, on calcium mobility (Table S1B). During oxidation of histamine (1.25 mM) with increasing concentrations of vDAO (5, 8, and 17 µM, as shown in Figure 4), increasing concentrations (7.4, 11.9, and 25.3 µM H₂O₂) were found (Table S1A). Separately, H₂O₂ was added at the same concentrations (as those produced by DAO) as the control (without histamine and without vDAO), measuring the calcium mobility (Table S1B). Despite the addition of H₂O₂ (control), calcium mobility remained unchanged at basal levels (Table S1B) as in the blank (no H₂O₂ added). These findings indicate that in our conditions of confocal microscopy the concentrations of H₂O₂ generated by vegetal DAO were insufficient to induce detectable calcium mobilization. Hydrogen peroxide is recognized as a redox signaling molecule able to, in eukaryotic cells, modulate intracellular calcium homeostasis by activating calcium-dependent signaling pathways [67]. The absence of a calcium response suggests that H₂O₂ accumulation may have been limited in our experimental system measuring the Fluo-4 AM fluorescence. A possible explanation can be the presence of endogenous catalase expressed by the differentiated Caco-2 cells. In fact, Baker & Baker (1992) showed an increase in catalase activity during differentiation of Caco-2 cells [68]. Since all our determinations were carried out with differentiated Caco-2 cells (day 21) the expressed catalase activity could explain the lack of response in terms of Ca²⁺ mobility in the presence of H₂O₂ alone added as a control. Thus, the modulation of Ca²⁺ mobility appears to be triggered by histamine and counteracted by DAO.

In terms of adverse effects, DAO commercial products are well tolerated and considered as safe, particularly at short-term administration. Most of them are designed as “food supplements” and contain DAO of pig kidney or legume sources.

However, an aspect to be taken into account is that DAO, in the presence of biogenic amines such as histamine, generates hydrogen peroxide (H₂O₂) a known damaging prooxidant [58]. When histamine was added to same Caco-2 cell line pretreated with vDAO only, a substantial decrease in viability (at about 40%) was observed. Conversely, when cells were pretreated with both DAO and Catalase, the observed viability was very high [58]. These previous results clearly indicated the damaging role of H₂O₂. This is why our proposed formulations for oral administration include DAO and Catalase [27,43]. Catalase not only protects cells and tissues against H₂O₂, but also releases oxygen in situ, shifting the reaction equilibrium in favor of the DAO oxidative decomposition of histamine or of other biogenic amines (i.e., tyramine) (Scheme 1). This bi-enzymatic DAO + Catalase formulation differs from the commercial products containing DAO only. The DAO and Catalase may be used long-term as food supplements, and even for prevention against histamine-related colorectal cancer [13].

DAO is very sensitive to acidic environments (i.e., stomach acidity). To prevent gastric degradation, DAO gastro-resistant formulations with adequate excipients such as Carboxymethyl-starch (CM-Starch) were reported [43].

Antihistamines are administered in order to alleviate local damages generated by secretion of histamine from activated mast cells and from neutrophil infiltrating cells [8,69].

The efficiency of DAO may suggest a substantial role as histaminase, but with the caveat that its action is mostly exerted at the intestinal level. It may act also as a support to antihistamine drugs, particularly in conditions of intestinal histaminosis such as food histaminosis or in cases of inhibition of intestinal DAO by the action of some drugs such as metformin [37].

The current in vitro investigation was conducted considering the substantial differences between antihistamine drugs and DAO histaminase activity. Antihistamine agents block receptors but histamine remains in the system with the possibility of acting on other receptors not blocked by the antihistamine drug. Conversely, DAO as a histaminase disrupts the histamine making it inactive. This aspect is important considering that Des acts specifically on the H1 receptor [9,70,71] leaving open the possibility of histamine to act on H2, H3, or H4 receptors or even on H1 receptors not fully blocked.

We believe that by decomposing histamine in the gut, DAO administration appears as a reinforcement but not a replacement of other medications including antihistamine and anti-inflammatory drugs. This study was conducted in vitro only and our data are not sufficient to support DAO as a clinical agent, but may help to clarify the role of Diamine Oxidase (DAO). The present data may contribute to understanding the role that diamine oxidase could play in future clinical development.

4. Conclusions

Vegetal DAO almost totally reduced the histamine-induced intracellular Ca²⁺ mobility. This efficiency of vDAO was higher (up to 96% inhibition) than that of Desloratadine (up to 72% inhibition). The DAO enzyme exerted this protective effect at a concentration 3-fold lower that of Des. This aspect is relevant for potential use of vDAO formulated for oral administration to prevent histamine-related dysfunction and reduce Ca²⁺ mobilization, thereby alleviating its effects.

Abbreviations

The following abbreviations are used in this manuscript:

AAP	aminoantipyrine
AUC	area under the curve
BSA	bovine serum albumine
Ca2+	calcium ion
Caco-2 cells	colorectal adenocarcinoma cells
CaCl2	calcium chloride
CI	confidence interval
DCHBS	sodium 3,5-dichloro-2-hydroxybenzenesulfonate
Des	desloratadine
DMEM	dulbecco’s modified Eagle essential minimum medium
EC50	half effective concentration
EDTA	ethylenediaminetetraacetic acid
RFU.	relative fluorescence unit
Fluo-4 AM	fluorophore-4 acetoxymethyl ester
FBS	fetal bovine serum
HR	histamine receptor
HEPES	4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid
Hist	histamine
HRP	horseradish peroxidase
HV	high voltage
H2O2	hydrogen peroxide
IC50	half inhibitory concentration
LC50	lethal concentration
Na2HPO4	dibasic sodium phosphate salt
NaH2PO4	monobasic sodium phosphate salt
SAO	serum amine oxidases
SDS-PAGE	sodium dodecyl-sulfate polyacrylamide gel electrophoresis
vDAO	vegetal diamine oxidase
λExc	excitation wavelength
λEm	emission wavelength

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/cells15131175/s1: Figure S1. DAO activity in different cell culture media. It was assayed in vitro with histamine as substrate at various concentrations in DMEM not supplemented (white) or supplemented (gray) with 15% fetal bovine serum (FBS) or in 100 mM sodium phosphate buffer pH 7.2 (black); n = 3 independent experiments (mean ± SD); Table S1. The effect of vDAO at various concentrations and of histamine on production of H₂O₂ and on Ca²⁺ mobility (A) versus the effect of H₂O₂ alone (control, at same concentrations) on Ca²⁺ mobility (B).

Author Contributions

Conceptualization, M.A.M., A.T.N., C.J., L.M. and P.P.; methodology, A.T.N.; software, A.T.N.; validation, M.A.M., C.J., L.M. and P.P.; formal analysis, M.A.M., A.T.N., C.J. and L.M.; investigation, M.A.M. and A.T.N.; resources, M.A.M., C.J., L.M. and P.P.; data curation, A.T.N. and M.A.M.; writing—original draft preparation, A.T.N.; writing—review and editing, M.A.M., C.J., L.M. and P.P.; visualization, A.T.N.; supervision, M.A.M.; project administration, M.A.M.; funding acquisition, M.A.M. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Informed consent was obtained from all subjects involved in the study.

Data Availability Statement

All data are contained within the article.

Conflicts of Interest

The authors declare no conflicts of interest.

Funding Statement

This research was funded by NSERC (Natural Sciences and Engineering Research Council), grant number 06912 (M. A. Mateescu). The project was also supported by a joint collaborative project (2017–2019) from the Ministère des Relations Internationales et de la Francophonie (MRIF) Québec, Canada; Ministry of Foreign Affairs and International Cooperation (MAECI) of Italy and Fondation Courtois. ATN was a holder of a doctoral scholarship from FRQS (Fonds de la Recherche du Québec-Santé), Québec, Canada, and of a Mobility Fellowship from CRIPA/FRQNT.