Effect of garlic consumption on the argyrophilic nucleolar organiser regions (AgNORs) in splenocytes and thymocytes of rats

Abstract

The number and size of nucleolar organiser regions (NORs) are related to cell proliferation. To test the hypothesis that lymphocyte proliferation is associated with garlic consumption, 17 6-week-old male Wistar rats were separated into two groups. For 30 days eight rats were given garlic solution by gavage while the remaining nine animals received distilled water. Silver staining of NORs was performed on the fixed tissues of spleens and thymuses. The nuclear area (NA), nuclear length (NL), total AgNORs area (TAA), total AgNORs length (TAL) and total AgNORs number (TAN) of 100 analyzable nuclei of lymphocytes from each tissue were recorded. In spite of a decline in the average NA and NL of thymocytes from garlic-treated rats (p<0.001), TAA and TAL increased significantly (p<0.001) in the splenocytes and thymocytes of this group. In conclusion, garlic may enhance the lymphocyte proliferation in these two important organs from the rat immune system.

Keywords:

• garlic
• argyrophilic nucleolar organiser regions
• lymphocyte proliferation
• splenocytes
• thymocytes

Introduction

Garlic (Allium sativum L.) is known as a potent medicine with broad therapeutic properties like antimicrobial, anti-inflammatory, anticancer and is used throughout the world (Colic, Vucevic, Kilibarda, Radicevic, & Savic, 2002; Ishikawa et al., 2006; Zamani, Vahidinia, & Ghannad, 2009). The recorded use of garlic in treatment of tumours dates as far back as 1550 BC. In the 1950s Weisberger and Pensky (1958) demonstrated in vitro and in vivo that thiosulfinate extracts of garlic inhibited the growth of malignant cells. Since then, garlic has been demonstrated in epidemiologic studies to be associated with a reduced risk of cancers (Ishikawa et al., 2006; Lamm & Riggs, 2001; Weisberger & Pensky, 1958).

The anticancer effect of garlic appears to be mediated by a number of mechanisms including antioxidant activity (Zamani, Ghiasvand, Hadei, Babaahmadi-Rezaei, & Pishdadian, 2009), direct inhibition of cancer cell growth (Lamm & Riggs, 2001) and potentiation of the immune system (Colic & Savic, 2000; Ishikawa et al., 2006; Liu, Su, Lii, & Sheen, 2009; Morioka, Sze, Morton, & Irie, 1993; Zamani et al., 2009). Up to now, most studies have examined the effect of garlic on proliferation of lymphocytes in cultures stimulated with a mitogen, whilst the in vivo effect of garlic on lymphocyte proliferation has received little attention (Colic et al., 2002; Liu et al., 2009; Morioka et al., 1993; Romano et al., 1997; Salman, Bergman, Bessler, Punsky, & Djaldetti, 1999).

The argyrophilic nucleolar organiser regions (AgNORs) are loops of chromosomal DNA 13–15 and 21, 22 in association with argrophilic staining proteins that are responsible for ribosomal RNA (rRNA) transcription (Sivridis & Sims, 1990). On the other hand, the close functional association between cell proliferation and the rate of rRNA transcription and synthesis means that with the increase in the number and size of AgNORs, the cell proliferation would also be high (Mello, Vidal, Russo, Planding, & Schenck, 2008; Mondal, Ghosh, & Ray, 2010).

Therefore, to test the hypothesis that garlic consumption is associated with lymphocyte proliferation we examined the AgNORs staining profile in the spleen and thymus of rats treated with garlic.

Materials and methods

Animals

Seventeen 6-week-old male Wistar rats were obtained from the animal facilities of Razi Institute, Tehran, Iran. Rats were housed in four plastic cages in an air-conditioned room, temperature 21–22 °C and humidity 55–65%. After 1 week of adaptation, the animals were randomly separated into two groups. Eight rats were given garlic solution in 4 ml distilled water (600 mg/kg) by gavage and nine rats received distilled water for 30 days. The animals were sacrificed under Thiopentone (50 mg/kg) anesthesia. Spleens and thymuses were then aseptically removed between 9 a.m. and 10 a.m. and washed three times in Hanks’ solution (Zamani et al., 2009).

Garlic

Garlic powder was obtained from Grandis Company, Hamadan, Iran. A mount of 600 mg/kg of garlic powder was mixed in 4 ml of distilled water and prepared fresh daily (Jastrzebski et al., 2007; Shua et al., 2008).

AgNORs staining

The spleens and thymuses were chopped and fixed in Bouin's solution (70 ml picric acid, 25 ml formaldehyde and 5 ml glacial acetic acid) for one week. The fixed samples were embedded in paraffin blocks and 3 µm sections were prepared using a microtome (Shandon, France). After tissue sections were deparaffinised and rehydratated, silver (AgNO₃) staining of NORs-associated proteins was performed as described by Derenzini and Ploton (1991). Briefly, the samples were treated with a solution containing 2 volumes 50% aqueous silver nitrate and 1 volume 2% gelatin in 1% aqueous formic acid (v/v). The aqueous solutions were prepared with deionised water. The optimal staining time was found to be 12 min at room temperature. Immediately after silver staining, the material was rinsed in deionised water, air-dried, cleared in xylene and mounted in Canada balsam (Figure 1).

Figure 1.  Silver-stained NORs pattern in thymocyte nuclei. Different sizes of nuclei were observed and stained pale red whereas silver stained NORs appeared as small black and dark brown intra nuclear dots in tissues from garlic-treated rats. Nuclei contain more than one dot (magnification = 1200×).

Image analysis

A NOVEX-B light microscope (Euromex, Holland) equipped with a high-resolution lens was used for image acquisition. One hundred analyzable and random nuclear images of lymphocytes in the white pulp of spleen and thymus samples were captured using a CMEX-1 USB-2 digital microscope camera (Euromex, Holland). Images were saved in TIFF format with the same size and resolution. Scion Image Software version Beta 4.0.2 (Scion Corporation, USA) was used to calculate nuclear area (NA), nuclear length (NL), total AgNORs area (TAA), total AgNORs length (TAL) and total AgNORs number (TAN). The calculated values were presented as pixels that had values from 0 to 255. According to this, white represented a value of 255, while black represented 0 (Cucer et al., 2007).

Statistical analysis

Results are expressed as mean and median±standard error. Student's t-test for arithmetic means and Mann–Whitney for medians were used to compare the examined groups. Correlation tests for the TAA and NA in two groups were also performed. All comparisons were two-sided, with p-values calculated for each group to indicate statistical significance. The statistical software used for this analysis was SPSS version 16.

Results

Results in Table 1 show that there were no significant differences between the mean mass of rats and spleens in the garlic-fed group compared with the control group. However, the mean mass of thymuses decreased significantly to 0.35±0.03 g in garlic treated rats compared to 0.45±0.02 g in the control group (p=0.017).

Table 1. Mass of rats, spleen and thymus in the control and the garlic-fed groups.

Mass	Controls (g)	No.	Garlic-fed (g)	No.	p value
Rats (pre treatment)	189.3±7.0*	9	188.1±7.1	8	0.906
Rats (post treatment)	281.3±16.1	9	274±10.1	8	0.722
Spleen	1.01±0.06	9	1.09±0.05	8	0.393
Thymus	0.45±0.02	9	0.35±0.03	8	0.017
Note: *Results are given as mean±SEM.

Two spleen samples (one from each of the two treatment groups), which contained undefined contours and inconspicuous AgNORs staining, were excluded from the analysis. In the splenocytes of both groups, smaller nuclei generally showed smaller AgNORs, whereas larger nuclei presented larger AgNORs aggregates. In thymic lymphocytes no correlation between TAA and NA was detected (Table 2).

Table 2. Correlation coefficient for the TAA and NA in different examined groups.

Cells	Groups	No. cells	Variable series	r	Significance
Splenocytes	Garlic	700	NA×TAA	0.122	p<0.01
	Control	800	NA×TAA	0.082	p<0.05
Thymocytes	Garlic	800	NA×TAA	0.010	p=0.771
	Control	900	NA×TAA	−0.046	p=0.160
Notes: NA, nuclear area; TAA, total AgNORs area.

In splenocytes, the arithmetic means of TAA and TAL were increased significantly (p<0.001) to 110.6±4 and 38.3±0.7 pixels in garlic treated rats compared with 83.4±3 and 32.5±0.6 pixels in the control group, respectively. TAA/NA in the garlic-fed animals was 0.13 compared with a decrease of 3% in the control group (TAA/NA ratio of 0.10) (Table 3).

Table 3. Image analysis of AgNORs staining in splenocytes.

	TAL pixel^2		TAA pixel^2		NL pixel^2		NA pixel^2
TAA/NA	Median	Mean	Median	Mean	Median	Mean	Median	Mean	TAN	No. splenocytes	No. spleen	Groups
0.13	33	38.3±0.7	75	110.6±4	115	116.0±1	862	896.6±14	900	700	7	Garlic
0.10	29	32.5±0.6	55	83.4±3	114	114.5±0.9	851	885.7±13	1002	800	8	Control
p<0.001	p<0.001	p<0.001	p<0.001	p<0.001	p=0.191	p=0.245	p=0.453	p=0.583	–	–	–	Significance
Student's t-test for arithmetic means and Mann–Whitney for medians were used to compare groups. NA, Nuclear Area; NL, Nuclear Length; TAA, Total AgNORs Area; TAL, Total AgNORs Length; TAN, Total AgNORs Number.

In thymocytes, the arithmetic means of NA and NL declined from 921.6±8.4 and 116±0.5 pixels in the control group, to 871.0±10 and 113.4±0.6 pixels in garlic-treated rats (p < 0.001), respectively. This contrasts with splenocytes, where the arithmetic means of TAA and TAL were increased significantly (p<0.001) to 108.1±3 and 39.4±0.7 pixels in the garlic-fed animals from 77.6±2 and 32.9±0.5 pixels in the controls, respectively. The TAA/NA ratio in the garlic-fed animals was 0.13 compared with an increase of 4% to a ratio of 0.9 in the control group (Table 4).

Table 4. Image analysis of AgNORs-staining in thymocytes.

	TAL pixel^2		TAA Pixel^2		NL Pixel^2		NA pixel^2
TAA/NA	Median	Mean	Median	Mean	Median	Mean	Median	Mean	TAN	No. thymocytes	No. thymus	Groups
0.13	33	39.4±0.7	72	108.1±3	112	113.4±0.6	828	871.0±10	1124	800	8	Garlic
0.09	29	32.9±0.5	56	77.6±2	116	116±0.5	895	921.6±8.4	1254	900	9	Control
p<0.001	p<0.001	p<0.001	p<0.001	p<0.001	p<0.001	p<0.001	p<0.001	p<0.001	–	–	–	Significance
Student's t-test for arithmetic means and Mann–Whitney for medians were used to compare groups. NA, Nuclear Area;NL, Nuclear Length; TAA, Total AgNORs Area; TAL, Total AgNORs Length; TAN, Total AgNORs Number.

Discussion

By evaluating the NA, NL, TAA, TAL and TAN in splenocytes and thymocytes of the control and the garlic-fed groups we observed that garlic enhanced the nucleolar activity in the lymphocytes of these two important immune system organs. This stimulatory effect was stronger in thymocytes compared with splenocytes, and nuclei sizes were smaller in thymocytes due to increased cell proliferation. To our knowledge, this is the first report describing the ability of garlic to increase in vivo lymphocyte proliferation in the absence of a mitogen.

Our results are in general agreement with the literature. Studies have already shown that garlic extracts are able to induce proliferation of lymphocytes from rat cervical nodes (Liu et al., 2009), cultured lymphocytes (Colic et al., 2002), human peripheral blood monocytes (PBMC; Salman et al., 1999), rat splenocytes (Colic & Savic, 2000), and human peripheral blood lymphocytes (Morioka et al., 1993). However, each of these previous studies also involved co-stimulation of immune cells with other factors such as Concanavalin A (Con A; Colic et al., 2002; Liu et al., 2009), pokeweed mitogen (Salman et al., 1999), phorbol myristate and the Ca ionophore (A23187) or R73 mononclonal antibody (Colic & Savic, 2000), and IL-2 (Morioka et al., 1993).

However, our results differ from other studies where administering aged garlic extract (AGE) to patients with advanced cancer of the digestive system improved NK cell activity, but caused no improvement in T-lymphocyte proliferation (Ishikawa et al., 2006). AGE alone was not mitogenic for rat thymocytes and splenocytes in culture, but higher concentrations of the extracts showed inhibitory effects, whereas lower concentrations significantly augmented proliferation of lymphocytes in cultures stimulated with Con A. The stimulatory effect of AGE has also been shown to be stronger in splenocytes (Colic et al., 2002). Ajoene, a compound originally isolated from ethanolic extracts of garlic, strongly inhibits the proliferation induced in human lymphocytes by the mitogens phytohemagglutinin (PHA), phorbol myristate acetate (PMA) and anti-CD3, and the capping formation induced in B lymphocytes by anti-IgM antibodies (Romano et al., 1997). Con A-induced human PBMC proliferation decreases following incubation with alliin (Salman et al., 1999), and aqueous extracts prepared from a garlic powder had no effect on rat peripheral blood lymphocytes after one month of administration by gavage (Zamani et al., 2009).

The results of this study are in contrast to the well-recognised antiproliferative effects of garlic in several tumour cells (Ishikawa et al., 2006; Lamm & Riggs, 2001; Pinto & Rivlin, 2001; Rivlin, 2009). This may be due to the metabolic differences between tumoural and normal cells (Velasco-Velazquez et al., 2006).

Up to now, more than 200 different biologically-active substances have been isolated from garlic. Among them, organosulfur compounds such as allicin, ajoene, diallyl trisulfide (DATS) or S-allylcysteine have the most potent biological activities (Colic et al., 2002). Feng et al. demonstrated that DATS had a dual role on T-lymphocyte proliferation in mice. At higher concentration (50 µg/ml) DATS inhibited T-cell proliferation triggered by Con A, but at lower concentrations (3–12.5 µg/ml) this substance augmented the proliferative response of T-cells to Con A (Feng et al., 1994). DATS and perhaps other allicin degradation products are the main candidates for stimulatory activity of the extracts and the effect of garlic may correlate with an increase in IL-2 production in lymphocytes.

Consumption of garlic and garlic-derived compounds has been shown to alter the rate of metabolism of testosterone through regulation of the cytochrome p450 enzymes. Garlic seems to inhibit the enzyme cytochrome p450, and the high level of steroids that results can cause weight reduction of thymus, but no effect on the spleen mass (Pinto & Rivlin, 2001; Zamani et al., 2009).

In conclusion, the results of this study indicate that the garlic aqueous solution induces enhancement in the nucleolar activity, and lymphocyte proliferation in the spleen and thymus of rats.

Acknowledgements

This study was funded by the research deputy of Hamadan University of Medical Sciences and approved by its ethical committee. The authors wish to thank Dr Brett Baillie (Australian National University, Australia) for reading the manuscript. The authors have no conflict of interest.