Multi Floral Honey Has a Protective Effect against Mammary Cancer Induced by 7,12-Dimethylbenz(a)anthracene in Sprague Dawley Rats

This research was conducted to study the protective effect of bee honey on the 7,12-dimethylbenz(α)anthracene (DMBA)induced breast cancer in rat model. The study consisted of three groups: honey group, positive control group (PC), and negative control group (NC) to which the carcinogen was not administered. All rats were fed the diet recommended by the American Institute of Nutrition for growing rats (AIN-93G), with addition of honey (50 g/kg diet) to the honey group. All Rats were fed their diets ad libitum on 12 hours dark/light cycle. At the age of 50 days all rats in the honey and PC groups were gavaged once by the carcinogen DMBA with a dose of 80 mg/kg body Wt. After three weeks of carcinogen administration, rats were palpated weekly to detect any tumor growth. After 18 weeks, all rats were sacrificed. The palpable structures and the mammary glands along with associated lymph nodes were removed and fixed in saline formalin and prepared for histopathological examination. The results revealed that the honey group diet significantly (p < 0.05) reduced the incidence rate of mammary cancer, palpable tumor multiplicity, tumor size and weight compared to the PC group. In conclusion, multi floral honey has a protective effect against DMBAinduced mammary cancer in the initiation, promotion, and progression stages of DMBA-induced mammary carcinogenesis. However, further research is needed to reveal the mechanisms that might have contributed to the preventive effect of honey against mammary cancer.

which exert beneficial effects against tumor cell proliferation (Tonks, Cooper, Price, Molan, & Jones, 2001). Due to its strong antioxidant property, honey may have the potential to prevent the development of cancer, as free radicals and oxidative stress play a significant role in the induction of cancers (Valko et al., 2007). Additionally, honey was reported to be a natural immune booster, natural anti-inflammatory agent, natural antimicrobial agent, natural promoter for healing chronic ulcers and wounds, and natural cancer vaccine (Othman, 2012). The carcinogen 7,12-dimethylbenz(α)anthracene (DMBA) is one of the two most commonly used substances to study mammary cancer in rodents (Guzman et al., 1999). Therefore, the aim of this experiment was to study the effect of honey on the incidence of breast cancer in rat model in which cancer was induced by (DMBA) through examining their effects on the multiplicity of tumors, tumor size, and tumor latency.

Animals
Thirty nine weanling female Sprague Dawley rats, three weeks of age (40-50 g), were used in the study. They were purchased from the Animal House of Jordan University of Science and Technology and were transferred to the animal unit/Department of Nutrition and Food Technology/Faculty of Agriculture/The University of Jordan. They were placed at a controlled temperature of 23±2 o C, humidity of 49±5%, and 12-hour light-dark cycle. The rats were divided into three experimental groups 13 rats in each: honey group, positive control group (PC), and negative control group (NC).

Preparation of the Experimental Diets
Animals were fed isocaloric and isonitrogenous diet which was prepared according to the Guidelines of the American Institute of Nutrition 1993 for growing animals (AIN-93 G), recommended by Reeves (1997) (Table 1) with some modifications as follows: 1) Soybean oil was replaced by an oil mixture that consists of canola oil and sunflower oil in a ratio of 0.7:1.0 (Al-Sayyed & Takruri, 2016), since soybean has been documented to be protective against breast cancer.
2) Multifloral honey was added to the honey group diet in a concentration of 50 g/kg diet. For this group we added only 50 g of sucrose instead of 100 g to have an isocaloric diet. Note. TBHQ: Tertiary Butylatedhydroquinone.

Preparation and Administration of Carcinogen
DMBA carcinogen (Santa Cruz Biotechnology/USA, purity ≥ 98%) was prepared by dissolving it in acetone and suspending it in sesame oil as described by Daniel and Richard (1964) and Lin et al. (2008). Then acetone was evaporated gently under liquid nitrogen.
At 50 days of age rats in the PC and honey groups (but not the NC group) were gavaged with a single dose of the sesame oil containing DMBA in a concentration of 80 mg/kg BW. After three weeks of carcinogen

Inciden
Results of rats are inv  NC group (13) 0 (0%) a 0 (0%) a PC group (13) 4 (30.8%) b 7 (53.9%) c Honey group (13) 0 (0%) a 4 (30.8%) b Note. 1 Data expressed as number of cases and percentages, values with different litters within a column differ significantly at (p < 0.05) using Fischer test; 2 n: number of rats used within each experimental group; 3 Incidence of invasive mammary carcinoma; 4 Adenosis: pro-carcinogenic or initiation stage. Table 3 shows the multiplicity of IMC (expressed as total number of IMC/group) among the experimental groups.

Multiplicity of IMC, Site and Latency
Only the PC group rats had high multiplicity of IMC (4 rats) whereas the NC and honey groups did not have any IMC as they did not have any tumors.  Note. 1 n: number of rats used within each experimental group; 2 multiplicity of invasive mammary carcinoma; 3 Number of days after carcinogen administration till the onset of the palpable tumor, expressed as mean day ± SEM. Table 4 shows that the largest average diameter of palpable tumors in the PC group was (20.81±4.18 mm) and that the average tumor weight (expressed as g/tumor/ tumor bearing rat) in this control group was (3.23±1.58 g). Note. 1 n: number of rats used within each experimental group; 2 multiplicity of invasive mammary carcinoma; 3 Values of size of tumor and its weight are expressed as mean ± SEM.  Note. BPS: benign palpable structure, data are expressed as total number of BPS/group and% from the number of animals in the group; 2 n: number of rats used within each experimental group.

Incidence of Malignant Tumors and Adenosis among the Experimental Groups
The incidence of IMC, which represents the malignant tumors, occurred only in the PC group rats (4, 30.8%).
The results of this study also showed that the honey group had no incidence of IMC which suggests that honey has protective effect against mammary cancer in rats. Several studies reported that honey has a strong antioxidant capacity (Yao, Datta, & Tomas-Barberan, 2003;Lurlina, Saiz, Fritz, & Manrique, 2009). These antioxidant effects of honey have the potential to prevent the development of cancer (Valko et al., 2007). Honey is rich in phytochemicals such as phenolic acids and polyphenols. The different polyphenols in honey were reported to have antiproliferative effects against several types of cancers (Jaganathan & Mandal, 2009).
These results are in agreement with those obtained in a Malaysian study, in which it was found that Tualang honey positively modulated the progression of DMBA-induced breast cancer in rats. Tumors were much less in number, volume, and weight in honey-treated rats compared to rats in the control group (AbdKadir, Sulaiman, Yahya, & Othman, 2013). Phenolic compounds, such as quercetin, were reported to induce apoptotic effects through estrogen receptors alpha and beta dependent mechanisms (Bulzomi et al., 2012). It is noteworthy that the amount of quercetin in honey is relatively high, making up 43 μg/100 g honey (Kurtagic, Redzig, Memic, & Sulejmanovic, 2013). Fauzi, Norazmi, and Yaacop (2011) mentioned that honey has cytotoxic effects in the human breast cancer cell lines MCF-7, and these effects were not shown in normal breast cell lines MCF-10 A. This suggests that the cytotoxic effects of honey are selective and specific to the breast cancer cell line only unlike most of anticancer drugs. Therefore, flavonoids and phenolic compounds in honey seem to be responsible for the cytotoxic effects on cancer cells (Erejuwa, Sulaiman, & Wahab, 2014).
Antimetastatic, antiproliferative, and anticancer effects of honey against breast cancer in rodents were also reported in other studies. Honey flavonoids particularly chrysin was shown to have antimetastatic effects against human breast cancer cells (Yang et al., 2013). Also it was reported that the anticarcinogenic activity of honey is due to its antiestrogenic effects and its potential in inducing mitochondrial membrane depolarization and apoptosis in breast cancer cells (Erejuwa et al., 2014). It was mentioned that the higher the phenolic content in honey, the greater its antitumor effects are (Jaganathan, Mondhe, Wani, Pal, & Mandal, 2010).
The histopathological examination in this experiment revealed an incidence of adenosis in the mammary tissues of the rats. The American Cancer Society ACS (2015) describes adenosis as an enlargement of breast lobules which contain more glands than usual. Other names of adenosis include: aggregate adenosis, tumoral adenosis, or adenosis tumor. However, adenosis is not a cancer, but can be developed later into a cancer. It is an early stage of the carcinogenesis process and keeping the tumor in the initiation stage.
The enlarged lobules may be distorted by a scar-like fibrous tissue in a condition known as sclerosing adenosis (SA) which is a special type of adenosis. In humans, having an SA increases the woman's risk for having breast cancer by two fold (ACS, 2015). SA is considered as an important entity for clinicians, because it mimics breast carcinoma clinically and on mammography. Some studies reported that SA represents a proliferative process which can lead to carcinomatous changes (Azzopardi, 1979).
The results of this study showed that the percentage of adenosis cases was in the PC group (58.3%), followed by the honey group (30.8). It has been reported that spontaneous tumors of different types and different body sites could happen in female Sprague Dawley rats with age, even with no induced carcinogenesis (Morii & Fujii, 1973). However the NC group rats in our study did not develop any tumor or even adenosis probably because of the short period of the experiment (

Multiplicity of IMC and Tumor Latency
The progression stage of carcinogenesis is characterized by further metabolism of the carcinogen which leads to additional lesions, malignancy, and growth of the tumors (Tsuda et al., 2004).Tumor growth can occur as a result of the proliferation of cancer cells (Ju et al., 2001). The results of this study showed that there was tumor multiplicity in 4 rats of the PC group, whereas the other experimental groups (negative control and honey groups) did not develop any cancer growth.
Tumor latency represents the period between DMBA administration and the appearance of palpable tumors. Latency period in DMBA-induced mammary cancer is a function of the promotion stage of carcinogenesis (Hieger, 1961). The promotion stage represents the time period starting after carcinogen administration. This period is characterized by the growth of mammary tumors (Welcsh, 1992). The delay in the appearance of palpable tumors indicates lower growth rate of tumor (Kodell & Chen, 2001). Also, this period is characterized by a loss of cell differentiation and disordered cell growth (Tsuda et al., 2004). In this experiment tumor latency for the PC group was 54.33±10.81 days, which is similar to the results obtained by Al-Sayyed, Takruri, and Shomaf (2014) who reported a latency period of 50 days in Sprague Dawley rats in which cancer was induced by DMBA.

Incidence and Multiplicity of Benign Palpable Structures among the Experimental Groups
The incidence of benign palpable structures (BPS) was reported by several researchers. They reported that a single intragastric dose of DMBA could induce benign mammary tumors in SD female rats (Noble & Cutts, 1959;Dias et al., 1999). Dias et al. (1999) documented an incidence rate of 34% for the benign mammary tumors after a single dose of 65 mg of DMBA administered into female Wistar rats. The variation in the incidence rates and multiplicities of BPS among different studies might be related to experimental conditions. In the present study, Sprague Dawley rats were used, the DMBA dose was 80 mg and the experimental period was 18 weeks only, whereas the experimental period in the Dias etal study was 24 weeks. Mann, Boorman, Lollini, Mc Martin, and Goodman, (1996) reported that there are three types of benign neoplasms in rat mammary glands including: fibroadenoma, fibroma, and adenoma. The most common benign neoplasms that occur in rat mammary gland is fibroadenoma. This type of neoplasm consists of both connective tissue and mammary epithelial cells. The proportion of these two cell types varies considerably from one tumor to another. Some tumors consist mainly of epithelial cells, whereas others are composed almost entirely of connective tissue.
Fibroma is another type of benign neoplasms in which the tumors are composed entirely of collagenous connective tissue without any epithelial component and which arise in the subcutaneous regions. The third type of benign neoplasms in rat mammary gland is adenoma. Tumors which represent this type are composed almost entirely of glandular epithelial structures with a scant connective tissue stroma. The epithelium is usually uniform with a cuboidal to columnar shape (Mann et al., 1996).
Abd El-Aziz et al. (2005) mentioned that mammary carcinogenesis can lead to hormonal disturbances, and accordingly, DMBA may induce hormonal disturbances. These disturbances might have led into the development of BPS, but not to adinocarcinoma. Other researchers reported that the incidence of BPS might be attributed to endocrine influences, particularly the estrogen hormone (Nobel & Cutts, 1959;Santner, Pauley, Tait, Kasenta, & Santen, 1997). In the present study, the incidence of BPS occurred only in the honey group which might indicate a protective effect of honey against carcinosis.

Conclusions
Based on the results of this study, it can be concluded that multifloral honey has protective effects against DMBA-induced mammary cancer in the initiation, promotion, and progression stages of DMBA-induced mammary carcinogenesis, as indicated by incidence rate, tumor latency, multiplicity and size.This effect was probably due to the strong antioxidants activity of honey.