Investigation of the effect of metoclopramide on proliferation signal molecules in breast tissue

Abstract

Metoclopramide (MCP) is a drug that has been widely used in recent years due to its hyperprolactinaemia effect on mothers during breastfeeding. The aim of this study was to investigate the proliferative changes that MCP may cause in the maternal breast tissue. In this study, 18 Wistar albino young–adult breastfeeding mothers with their offspring were divided into three groups: control group, low‐dose MCP‐applied group and high‐dose MCP‐applied group. The experiment was carried out during the lactation period and at the end of 21 days. Prolactin, BrdU and Ki‐67 breast tissue distributions were evaluated by immunohistochemistry, and tissue levels were evaluated biochemically by the ELISA method. According to ELISA and immunohistochemistry results in breast tissue, there was no significant difference between Ki‐67 and BrdU results in all groups. Metoclopramide did not change the expression of proliferation molecules Ki‐67 and BrdU in breast tissue. These results suggested that while metoclopramide increases breast proliferation, it does not have the risk of transforming the tissue into a tumour.

1. INTRODUCTION

Breastmilk is still considered to be an excellent source of nutrition for all newborns today. ¹ Breastfeeding has advantages for both maternal and child health. These advantages include health, nutrition, development, and psychological, immunological and economic benefits. ² However, fatigue, anxiety, stress and various diseases can cause a decrease in breastmilk and malnutrition in infants. ³ Therefore, women with insufficient milk production may resort to herbal and pharmaceutical galactagogues to increase milk production. ⁴

Galactagogues are drugs that help initiate and maintain adequate milk production. Many exhibit pharmacological effects by interacting with dopamine receptors, resulting in increased prolactin levels; that is, the amount of milk increases. ⁵ Metoclopramide (Metpamid), sulpiride (Zegerid), domperidone (Motilium), chlorpromazine (Largactil) and exogenous hormones (TRH, growth hormone) are the main galactagogues. ⁶ , ⁷ Metoclopramide (MCP) increases the prolactin level by showing an antagonistic effect against dopamine release in the central nervous system. In addition, since there is a dopamine receptor (D2‐R) antagonist in the central and peripheral nervous system, it is used as an anti‐emetic in the treatment of gastroesophageal reflux disease by stimulating upper gastrointestinal motility and in the treatment of many diseases. ⁸ , ⁹

Molecular studies in the literature on the side effects of MCP use during breastfeeding in newborns and mothers are insufficient. MCP easily crosses the blood–brain barrier, causing fatigue, headache, anxiety, intestinal disorders, especially depression, and extrapyramidal side effects. ¹⁰ , ¹¹ , ¹² , ¹³ , ¹⁴

In our study, we aim to investigate the immunohistochemical and biochemical analyses of proliferative changes in breast tissues of mothers with metoclopramide administered to rats during lactation. For this purpose, the distributions and levels of prolactin, BrdU and Ki‐67 expressions in tissue homogenate have been evaluated.

2. # MATERIALS AND METHODS

2.1. Ethical Approval

For experiment protocol, ethics approval was obtained from the Animal Experiments Local Ethics Committee, Manisa Celal Bayar University, with the date and protocol no. 21.01.2015/77.637.435.08.

2.2. Experimental Protocol

In this study, 18 Wistar albino young–adult breastfeeding mother rats were used. Rats with their offspring were divided into three groups (n = 6). Group 1: healthy control; Group 2: low‐dose MCP‐applied group (10 mg/kg 21 days, 2 doses per day, intraperitoneally (i.p.)) ¹⁵ ; and Group 3: high‐dose MCP‐applied group (45 mg/kg 21 days, 2 doses per day, ip). ¹⁶ In this study, the MCP was obtained from Sigma‐Aldrich (metoclopramide hydrochloride, CAS No: 7232‐21‐5). During lactation, mother rats and their offspring were kept in the same cage and no treatment was given to the offspring. MCP was injected intraperitoneally to mother rats, and hyperprolactinaemia was induced. The experiment continued throughout the lactation period, and 21 days later, the rats were killed by intraperitoneal injection of ketamine (90 mg/kg) and xylazine (5 mg/kg). Right breast tissues removed after cervical dislocation were placed in fixatives for immunohistochemical analysis, and left breast tissues were placed in tissue extraction buffer for biochemical analysis. In the study, tissue distributions of prolactin, BrdU and Ki‐67 were evaluated by immunohistochemistry, and tissue levels were evaluated biochemically by the ELISA method.

2.3. Immunohistochemistry and histochemistry

For immunohistochemical studies, sections (5 µm) were deparaffinized in xylene and rehydrated through reduced alcohol series, then washed with distilled water for 10 min. They were incubated in 2% trypsin (Sigma‐Aldrich) in the Tris buffer (50 mM Tris base and 150 mM NaCl) at 37°C for 15 min to release the antigenic sites. To suppress endogenous peroxidase activity, the sections were kept in blocking solution (Invitrogen) for 10 min. after being incubated at 3% of H₂O₂ for 15 min. Sections were then incubated with prolactin (goat polyclonal), BrdU (mouse monoclonal, Thermo Scientific) and Ki‐67 (rabbit polyclonal, Thermo Scientific) for one hour at +4°C. Sections washed with PBS solution were incubated with biotinylated secondary antibody and streptavidin‐conjugated horseradish peroxidase for 20 min (Invitrogen). Diaminobenzidine (AEC, Thermo Scientific) was used to visualize the reaction. Background staining was done with Mayer's haematoxylin (72804E; Microm). Immunostaining scores were evaluated by two independent researchers, and photographs were evaluated using the CX31 light microscope (Olympus). In staining, at ×400 magnification, five areas were selected randomly in each preparation and the H score was calculated according to the intensity of the involvement and the percentage of the amount of involvement. Retention density was semi‐quantitatively scored as 0 (0, no involvement), 1 (+, weak immunoreactivity), 2 (+ +, moderate immunoreactivity) and 3 (+ + +, strong immunoreactivity). The amount of retention was scored as 1 (0% to 10% focal), 2 (11% to 50% regional) and 3 (51% to 100% diffuse) by dividing the cell structures consisting of per cent immunoreactivity to the total cell structure. The density and quantity scores obtained for each area were calculated with the Σ Pi formula. (i + 1) (Pi =percentage of retention, i = retention density). ¹⁷ The results were obtained, and a single value was reached for that slide. Tissues were stained with toluidine blue (Sigma‐Aldrich) for mast cell count in breast tissues. Mast cells were counted at 40 magnification by taking 10 sections from each tissue, and a single number was reached for each animal. The obtained data were evaluated with spss (Statistical Package for the Social Sciences) software 15.0.

2.4. Biochemistry

2.4.1. Preparation of rat tissue samples and protein measurements

Tissue homogenization was performed for biochemical analysis of rat tissue samples. Tissue extraction buffer prepared with a mixture of 100 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X‐100, 5% sodium deoxycholate and protease inhibitor cocktail was used for tissue homogenization. Tissue extraction buffer (w/v) of 1:10 was used for 0.1 g of each tissue. The resulting homogenates were then centrifuged at 4000 rpm for 15 min, and supernatants were taken for protein measurement and kept at −80°C until the day of analysis for biochemical studies. The bicinchoninic acid assay (BCA) protein measurement method was used for protein measurement in homogenates (Cat No: Thermo, BCA Protein Assay Kits, 23225), and some of the supernatants were kept at −80°C analysis day for biochemical studies.

2.4.2. Enzyme‐linked immunosorbent assay (ELISA)

Prolactin (Cusabio, CSB‐E06883h), BrdU (Sun Red Biotechnology, 201‐11‐0652) and Ki‐67 (Cusabio, CSB‐E13175r) levels in homogenized breast tissues were analysed with ELISA kits. All methods were performed in accordance with kit procedures. The absorbance was measured at 450 nm using Synergy HT, Multi‐Detection Microplate Reader and BioTek plate reader devices. Results are given as pg/mg protein. The data obtained from the statistical analysis research were evaluated in the spss (Statistical Package for the Social Sciences) software 15.0 program. The Kruskal–Wallis analysis was performed for comparisons between groups. The Mann–Whitney U test was used for pairwise comparisons of the groups. Data are given as mean ± standard deviation.

2.5. Statistical analysis

The data obtained from the research were analysed in the spss 15 package program for Windows. Histological and biochemical values, percentages and averages of the study groups were taken, and the Kruskal–Wallis analysis was performed with non‐parametric tests. The groups were compared within and among themselves. Pairwise comparisons of the groups were made with the Mann–Whitney U test.

3. RESULTS

3.1. ELISA results

There was no significant difference in breast tissue Ki‐67 and BrdU ELISA levels between all groups (p = .05) (Table 1). There was a statistically significant difference in prolactin tissue levels of all groups (p < .05) (Table 2).

TABLE 1.

Ki‐67 and BrdU breast tissue ELISA levels of mother rats in control, low‐dose and high‐dose groups

Biochemical analysis	Ki‐67	BrdU
	Mean ± SD	Mean ± SD
Control	147.19 ± 9.54	219.72 ± 85.16
Low MCP	148.95 ± 32.34	239.78 ± 76.73
High MCP	149.98 ± 20.62	348.07 ± 27.72

^* p < .05 (Kruskal–Wallis test)

TABLE 2.

ELISA and HSCOR prolactin levels in breast tissues of rats in control, low‐dose and high‐dose groups

HSCOR	Prolactin*	HSCOR Prolactin*
	Mean ± SD	Mean ± SD
Control	25.44 ± 3.13	140.00 ± 11.33
Low MCP	31.72 ± 0.58a	181.50 ± 5.61a
High MCP	37.13 ± 3.09b,c	218.50 ± 18.72b

^aSymbolizes the comparison between control and low dose, p < .05 (Mann–Whitney U test).

^bSymbolizes the comparison between control and high dose, p < .05 (Mann–Whitney U test).

^cSymbolizes the comparison between low dose and high dose, p < .05 (Mann–Whitney U test).

^* p < .05 (Kruskal–Wallis test).

3.2. Results of toluidine blue staining of mast cells in breast tissue

When the number of mast cells in the breast tissue was compared, a significant difference was observed between the groups. In the pairwise comparisons between the groups, it was observed that the low‐dose group was significantly higher than the control group (p < .05). No statistical difference was observed when the low‐dose group was compared with the high‐dose group (p = .05) (Figure 1) (Table 3).

FIGURE 1.

Breast tissue toluidine blue staining control (A), low‐dose metoclopramide (B) and high‐dose metoclopramide (C). : Mast cells (1) (×100), (2) (×400)

TABLE 3.

Mast cell amounts of breast tissues of mother rats in control, low‐dose and high‐dose groups

SCOR	Mast cell
	Mean ± SD
Control	17.66 ± 7.63
Low MCP	37.66 ± 6.08 a
High MCP	34.66 ± 8.47

^a Symbolizes the comparison with the control group, p < .05 (Mann–Whitney U test).

3.3. Results of immunohistochemistry

While moderate‐to‐severe prolactin expression was observed in the mammary gland epithelium in the control group (Figure 2A1), it was noted that the reaction increased in low‐ and high‐dose groups in epithelial cells and there was a very strong staining of cells in connective tissue (Figure 2A2 and A3) (Table 2). It was determined that some cells in the control, low‐dose and high‐dose groups were positively stained with Ki‐67 (Figure 3A1, B1 and C1). In BrdU immunostaining, a moderate expression was observed in mammary epithelial cells of mammary tissue of all three groups (Figure 3A2, B2, C2) (Table 4).

FIGURE 2.

Breast tissue prolactin immunostaining, control (A), low‐dose metoclopramide (B) and high‐dose metoclopramide (C). : breast epithelial cells showing prolactin positive; *: connective tissue cells, Mayer's haematoxylin counterstain (×400)

FIGURE 3.

Breast tissue Ki‐67 immunostaining (1) and BrdU immunostaining (2). Control (A), low‐dose metoclopramide (B) and high‐dose metoclopramide (C). : Breast epithelial cells showing Ki‐67‐positive immunoreaction; : breast epithelial cells showing BrdU‐positive immunoreaction, Mayer's haematoxylin counterstain (×400)

TABLE 4.

BrdU and Ki‐67 HSCOR levels of breast tissues of mother rats in control, low‐dose and high‐dose groups

HSCOR	Ki‐67	BrdU
	Mean ± SD	Mean ± SD
Control	95.50 ± 9.02	123.00 ± 3.74
Low MCP	103.00 ± 3.74	125.33 ± 7.00
High MCP	124.66 ± 4.32	124.66 ± 9.37

*p < .05 (Kruskal–Wallis test).

4. DISCUSSION

MCP causes hyperprolactinaemia by increasing prolactin level with its antagonist effect against dopamine release in the central nervous system. ⁷ In addition, metoclopramide is frequently preferred as a prokinetic agent ¹⁸ in the treatment of gastroesophageal reflux disease and as an anti‐emetic in pregnancy, as it is a dopamine receptor (D2‐R) antagonist in the central and peripheral nervous system. ¹⁹ , ²⁰ MCP can easily cross the blood–brain barrier and cause asthenia, drowsiness, extrapyramidal reactions and neuroendocrine effects (galactorrhoea). ²¹ , ²² , ²³ Despite these known side effects, metoclopramide is given to mothers during breastfeeding due to its prolactin‐increasing effect. It was observed that metoclopramide given to mothers in various doses increased prolactin ¹² in the mother, which has a positive effect on milk production, and increased prolactin, ⁷ TSH and FT4 ²⁴ in the newborn. In our study, the observation that metoclopramide causes hyperprolactinaemia in the tissue is similar to other studies. According to both our ELISA and immunohistochemical results, prolactin levels in the high‐dose group were higher than in the low‐dose and control groups.

Metoclopramide is known to pass into both milk and plasma. ¹⁴ Lewis et al measured the level of metoclopramide excreted in milk in 10 women in the study. All women were given a single dose of 10 mg oral metoclopramide for 7–10 days while breastfeeding after delivery, and blood and milk samples were measured 2 h after taking the dose. While metoclopramide was found to be 69 ± 30 ng/ml in maternal plasma, it was measured as 126 ± 42 ng/ml in breastmilk. ²⁵ In this study, it was shown that newborns whose mothers were given metoclopramide and who received 1 litre of milk per day passed through breastmilk at an amount of 45 μg/kg/day. ²⁵ It has also been reported in other studies. It is found in low concentration in milk and is pharmacologically effective. ¹⁴ , ²⁶ , ²⁷ The recommended dose of metoclopramide for galactagogue effect is 10–15 mg between 1 and 4 weeks. ¹⁶ According to some researchers, it was emphasized that metoclopramide administration in nursing mothers should not exceed 45 mg/kg. ²⁸ In our study, we used a low dose of metoclopramide 10 mg/kg for 3 weeks and 45 mg/kg.

In one study, mid‐temporary intestinal discomfort ²⁹ was not observed in mothers with MCP administration at high doses (30 and 45 mg/day), and no side effects were observed in another study. ²⁷ Among the side effects that may occur, fatigue, headache, anxiety and intestinal diseases were reported in the mother. ¹¹ According to Ehrenkranz et al, 10% of mothers reported irritability, lethargy and fatigue, headache, intestinal disorders and 1% extrapyramidal reactions. ¹⁰

Physiological changes occur due to circulating oestrogen, progesterone and prolactin hormones during pregnancy and lactation. These hormones have reproducing effects on the glandular and ductal tissues of the breast. It is also known that oestrogen and prolactin increase breast cancer growth. ³⁰ , ³¹ However, there is no study in the literature evaluating possible proliferative changes and dose differences in maternal breast tissue in experimental studies with MCP. Ki‐67 is a core protein seen in proliferating cells. It is predominantly seen in the G1, S, M and G2 phases. It is not found in the G0 phase. ³² It is a protein that shows the morphological features of cell proliferation and is frequently used in mitotic index and tumour grading. In general, there is a good correlation between Ki‐67 staining and mitosis. ³³ The percentage of cells showing positive nuclear staining by immunohistochemistry for Ki‐67 indicates the proliferation index. This rate is higher in aggressive tumours. It has been shown that high Ki‐67 ratio is a poor prognostic factor in many system tumours (breast, lung, oesophagus, kidney and prostate cancer, malignant melanoma, non‐Hodgkin lymphoma, glial tumours, etc). ³⁴ BrdU is a thymidine analogue. It is only used for indicating cells in S phase by labelling with anti‐BrdU antibody. ³⁵ , ³⁶ BrdU, which is a commonly used marker in immunohistochemical methods, is a thymidine analogue that participates in the structure of the DNA in the replication phase of the cycle of the cell to introduce new cells. ³⁷ The integration of S phase cells with BrdU can be readily determined by membrane permeabilization with anti‐BrdU‐specific antibodies.

In our study, there was no significant difference between Ki‐67 and BrdU results in all groups according to ELISA and immunohistochemical results in breast tissue. This leads to the thought that metoclopramide increases proliferation in the breast but does not have the risk of transforming into a tumour. Mast cells are known to have a role in the regulation of proliferative and inflammatory cells in the breast. ³⁸ We found a significant increase in mast cells in the low‐dose group. It showed that mast cells were associated with inflammatory cells, not proliferative cells. Therefore, in our future experimental study, it is seen that breast tissue should be examined for the expression of inflammation between groups.

Metoclopramide did not change the expression of Ki‐67 and BrdU proliferation molecules in breast tissue. As a result, while metoclopramide increases breast proliferation, it does not have the risk of transforming the tissue into a tumour.

CONFLICT OF INTEREST

The author(s) declared no potential conflicts of interest with respect to the research, authorship and/or publication of this article.

AUTHOR CONTRIBUTIONS

All authors designed the study, interpreted and analysed the data, and reviewed the manuscript. SGG and SİÇ conceptualized and designed the study. NU and GNY collected the data. SGG and GNY analysed and interpreted the data. NU drafted the article. SGG and NU gave final approval of the version to be published.

Umur N, İldan Çalım S, Yazıcı GN, Gurgen SG. Investigation of the effect of metoclopramide on proliferation signal molecules in breast tissue. Int J Exp Path. 2022;103:83–89. doi: 10.1111/iep.12433