Chemopreventive effect of Quercus infectoria galls on DMBA induced mouse skin tumorigenesis

Introduction

Living organisms are daily subjected to physiological and environmental chemical substances; in both developing and developed countries, carcinogenic substances associated with environmental pollution are regarded as important lethal factors that are threatening the public health. DMBA is not excluded from the environmental pollutants and it is greatly found in smoke of different sources including cigarette and car exhaust (1). DMBA is a potent carcinogenic agent involved in different steps of cancer development including initiation and promotion stages, it has been widely used as a cancer inducing agent in oncology studies (2). Scientists found that DMBA binds to DNA in mouse skin a target tissue, through a 1.2,3,4-ring dihydrodiol epoxide which links to and ruin DNA, Oxidative stress caused by Reactive Oxygen Species (ROS) that are greatly produced during metabolism of DMBA also found to play roles in developing cancer of skin (3). DMBA increases mutation rates of keratinocytes in skin epidermis and causes changes within composition of the cellular membrane, inactivate detoxifying enzymes and interfere with signaling proteins of cells, affect cells metabolism and deteriorate proteins structure (4). These lead to uncontrolled growth of cutaneous cells into squamous cell carcinoma (SCC), which regarded as the second most common type of epidermal neoplasia, the recently detected cancer globally classified as the skin tumor and up to 80% of these cancers found to be resulted from solar ultraviolet radiation. Squamous cell carcinoma (SCC) found to be induced directly by ultraviolet light damage, besides other causes like papilloma viruses from animal and human origin, existing ulcer and wounds, burns and also in old scars (5).

Recently, herbal treatments have been extensively considered in controlling and treatment aspects of skin cancer. Accordingly, identification of anti-cancer promoting agents within the medicinal plants would be of great importance in controlling of pathogenesis of skin cancer (6). Traditional medicine has obviously used medicinal plants, thus, in the current study, a plant specifically, Quercus infectoria (QI) galls has been chosen due to the fact that the plant has been historically widely involved in herbal treatments, it had been used for inflammatory diseases and wound infections (7). Tannins (gallotannic acid) is predominant constituent of Quercus infectoria galls up to 50-70%, the compound is well known for its properties as anti-oxidant, anti-mutant, and anti-cancer (8). Studies reported various medical values of Quercus infectoria galls as larvicidal (9), anti-inflammatory (10), antibacterial (11), anti-fungal (12), antioxidant (13), and antidiabetic (14). To the best of our knowledge, there aren’t any animal studies on the effect of QIGE in the prevention of skin carcinogenesis.

Hence, the current study aimed to assess the effectiveness of homogenized extract of Quercus infectoria galls in modifying the carcinogenic process in mice by measuring tumor incidence, tumor diameter, accumulating number of tumor, tumor burden, tumor yield, latent period and examination the histopathological changes in the DMBA / croton induced tumors in mice skin before and after Quercus infectoria galls aqueous extract (QIGE) administration.

Material and methods

Chemicals

 DMBA the initiator was supplied by from ACROS organics -Belgium, and croton oil (promoter) were procured from Alfa Aesar-USA. 100 mg of DMBA dissolved in acetone 100 ml, and then 1 ml of solution (single dose) applied on the shaved region of mouse skin. To give a solution of 1% dilution, croton oil was mixed in acetone. From local sources, other analytical grade chemicals were purchased.

Plant material

Healthy Mature galls of Quercus infectoria (Figure 1) was collected locally from Gara Mountain, Amedi, Duhok, Iraq in October 2017, the plant galls were thoroughly washed by tap water and at room temperature the galls were air-dried in doors. The plant is authenticated by the Professor Dr. Saliem E. Shahbaz an expert in College of Agricultural Engineering Sciences, University of Duhok, Iraq.

Figure 1: Quercus infectoria galls.

Preparation of the homogenized Quercus infectoria galls

Electric grinder was used to crush the cleaned galls into to a fine powder which was homogenized in distilled water for 24 hours. To prepared aqueous extract of plant, in distilled water 50 gm of plant powder were suspended in Erlenmeyer flask and by magnetic stirrer the suspension was stirred over night at 45˚C. Following 24h, the precipitants were filtered firstly by using gauze and followed by filter papers, the filtration processes were repeated 5 times and before using rotary evaporator to dry the pooled extract under reduced pressure at 45ºC (15). The obtained crude extract from the used powdered plant was weighted by the following formula; yield of extract % = weight of petri dish with extract - weight of empty Petri dish /50*100. The crude extract then was stored in deep freezer at -20˚C to be used later. For the following experiments the extract was dissolved in PBS (solvent) to prepare the experimental doses of 2 gm/kg b.w.

Experimental animals

Random-breed male Swiss albino mice (Mus musculus), strain Balb/c (25-30 gm, 8-10 weeks) were obtained from University of Duhok, College of Science animal house. At standard laboratory conditions, the used animals in this study were kept in well controlled climate at temperature 22-26°C, exposed to cycles of 12h dark /12h light. The standard locally prepared diets were fed to mice with water ad libttum. Before starting of the experiment, the used mice were acclimatized for a week.

Experimental design

To test the chemopreventive efficacy of QIGE against skin papillomagenesis in mouse induced by DMBA and croton oil, 75 mature male mice used in this study were chosen at resting-growth phase from the inbred colony and grouped into seven different groups (Figure 2), the dorsal skin was carefully shaved (2 cm diameter) by electric clipper to avoid injuries two days before application of the initiator. The groups were; group 1: Croton oil; n =10: these animals were treated with 1 ml of croton oil solution (1ml croton oil/100 ml acetone) applied cutaneous to the shaven part in treated mice (3 times /week) over 16 weeks, group 2: Acetone; n = 5: animals of this group were injected with 1 ml of an acetone on the dorsal clipped portion (3 times /week) over 16 weeks, group 3: DMBA (alone); n=10: individual dose DMBA (100 mg/100 ml of acetone) was injected to the cleaned shaven skin area of the used animals and left without treatment for a period of16 weeks, group 4: QIGE (-ve); n=5: animals were given Quercus infectoria galls extract orally (2 gm/kg. b.w. 3 times/week, following a period of 16 weeks, the treated rats were killed and their dorsal area of skin were taken for histopathological studies, group 5: DMBA+ Croton oil (+ve); n=15 mice: an individual dose of DMBA (100 mg DMBA /100 ml of acetone) injected to the shaven area of the skin in treated animals., croton oil 1% (1ml croton oil /100 ml acetone) applied after 14 days of DMBA application. The treatment with croton oil was trice weekly and continued up to the sixteenth week at alternate days, group 6: Pre-Treatment; n=15 mice: in this peri-initiation treatment group, animals were administered Quercus infectoria galls aqueous extract (2 gm/kg) orally for 7days, then DMBA solution single dose applied over the shaven part of skin, two weeks later mice were administered Quercus infectoria galls aqueous extract 2 gm/kg orally 3 times/week followed by applying 1% croton oil on the shaven part of the skin until the end of the experiment, group 7: Post-Treatment; n=15: In this post-initiation treatment group, a single dose of DMBA solution applied topically to the shaven part of the mice skin, then after two weeks mice were administered Quercus infectoria galls aqueous extract 2 gm/kg orally 3 times/week followed by applying 1% croton oil on the shaven part continued to 16 weeks, modified from (16).

In vivo determination of the chemo-modulatory effect of QIGE on skin carcinogenesis induced by DMBA/Croton oil

To determine the in vivo chemo-modulatory efficacy of QIG on cancer development in skin by carcinogen DMBA/Croton oil, the listed parameters were applied.

Morphological examination of tumor

After the completion (i.e. 16 weeks) of the experiments, the considered parameters are: the tumors number in skin of individual mouse was reported. Skin papilloma were described as lesions ˃1 mm in diameter and that were found minimally over two successive inspections (17). Tumor incidence: the number of affected mice with at least one lesion of skin cancer and referred as % incidence. Tumor yield: The average number of papilloma / mice. Tumor burden: The average number of tumors / tumors bearing mouse. Cumulative number of tumors: the total number of tumors found in all mice. Tumor Diameter: The diameter of each tumor scaled by electronic caliper. Average latent period: the required time-lag for the appearance of 50% of tumors following using of the promoting agent was detected; the formula ∑ (𝐹𝑋/𝑛) was used to determine the average latent period, which was determined by multiplying the tumors number observed per week by number of weeks following using promoting agent and divided by the total number of tumors. Where F is the reported numbers of tumors / week, X weeks numbers, and in the sum of tumors numbers. Measurement of body weight: The body weight of the individual animal was daily taken starting from the first date of the experiment and continued during the whole experiment time or was end by the animal death. Progressive increasing in body weight of the all mice groups was observed and recorded during treatment periods but their weight returned to nearly normal by the end of the experiment.

Figure 2: Dose application pattern in control and treatment groups.

Histological evaluation

For histopathological studies, skin tissues were taken from the affected part in treated rats; the taken tissues were fixed in 10% of formalin buffer solution then rinsed in water, a range of increasing concentration of ethanol (70% /24hrs, 80% /1hr, 90% /1hr and 100 % two exchanges, 1hr each) were used to dehydrate the tissues. Xylene was used for 10 min for twice to clear the samples. The samples were infiltrated in paraffin wax at 58ºCin oven for two exchanges 2 hours each. Paraffin block in the cassette were made for the samples which were cut into four parts before embedding the in wax. The embedded sections were cut at thickness of 4 µm and then by hematoxylin and eosin (H&E) stained following the staining protocol (18). The H&E stained sections were examined microscopically using light microscope (Olympus VANOX-S Germany), images were taken by digital camera. The slides were evaluated by an expert pathologist who was blind to data. At least 10 fields per slide were examined and scored.

Statistical analysis

One-way analysis of variance (ANOVA) test was used to analysis the data of the present study. Duncan multiple range test was used to determine the specific variations between the groups and SPSS (V.23) was used to evaluated the results. P ˂ 0.05 was the accepted significant level. Values were presented as mean ± standard error of the mean (SEM) of 5-15 mice/group.

Results

Effect of QIGE on changes in body weight of mice

Progressive increases in the body weight of the treated mice after initial treatment were reported and such weight was found to show changes significantly in the body weight during the experimental protocol (Table 1).

Effect of QIGE on morphological examination of tumor

At 16th week of DMBA/Croton oil-treatment, the incidence of tumors developed papilloma (93% incidence). The oral pre initiation treatment and post initiation treatment of QIGE at doses of 2 gm/kg significantly reduced the incidence of papilloma, and the recorded incidence in these groups was counted 0.40±0.131 and 0.533±0.133 respectively (Table 2).

Table 1: Variations in body weight during skin carcinogenesis induced by chemical in mice with or without Quercus infectoria galls aqueous extract

The data were presented as mean ± SEM (n=5-15). QIGE: Quercus infectoria galls aqueous extract; -ve: Negative control; DMBA: 7,12-Dimethylbenzanthracene; +ve: positive control. The different letters in the column referred to the significant differences at level (P˂0.05).

Table 2: Effect of Quercus infectoria galls aqueous extract on morphological appearance of tumor

The data were presented as mean ± SEM (n=5-15). QIGE: Quercus infectoria galls aqueous extract; -ve: Negative control; DMBA: 7,12-Dimethylben(z) anthracene; +ve: positive control. The different letters in the column referred to the significant differences at level (P˂0.05).

Oral administration of QIGE in group 6 (pre-initiation) and group 7 (Post-initiation) significantly reduced (P<0.05) the accumulating number of tumors (total number of papilloma until the finish of experiment) 0.92±0.21 and 1.21±0.28 respectively as compared to the positive control group5 (+ve) 3.07±0.69 (Table 2). Concerning the tumor diameter, the groups 6 and 7 did not show significant (P<0.05) decreasing in tumor dimeter in comparison to mice were treated with DMBA/Croton oil (Table 2). While the average latent period (the required for developing of 50% of tumors after application of the promoter) was considerably longer in group 6 (Peri-initiation) and 7 (Post-initiation) i.e. 12.3±0.003 weeks and 9.7±0.01 weeks, respectively, while it was found higher in treated control with the carcinogen (Group 5) i.e. 7.3±0.03 weeks. Significant reductions in tumor yield and tumor burden were reported in the animals of group 6 and 7 to 0.83±0.01, 1.20±0.01 and 1.20±0.01,1.69±0.01 respectively comparing with their positive groups. None of the mice from the group 1(croton oil), group 2, the vehicle-treated group (acetone alone), group 3 (DMBA alone), and group 4 (QIGE alone) developed tumors (Table 2). Moreover, redness and inflammation were observed in mice skin of treated groups 1, 2, 3 and hair loss was observed in skin of group 1 and 2 while group 3 its hair falls partially, and group 4 its hair did not fall.

Figure 3: Histopathology observation in skin tissue of experimental animals: (A): croton oil treated mice group; (B): acetone (alone) group vehicle treated mice; (C): DMBA (alone) group; (D): QIGE (-ve control) group (2 mg/kg); (E). DMBA and croton oil (+ve control) group; (F): QIGE, DMBA and croton oil (pretreat.); (G): DMBA, croton oil and QIGE (post-treat.) group. Abbreviation: E, epidermis; D, dermis; HD, hypodermal; PC, pearls cells; iPC, initial pearls cells; KP, keratinized pearls; H, hyperkeratosis; DK, dyskeratosis; P, papilloma; iP, initial papilloma EC, epidermis cells; I, Inflammation; De, degenerative; N, Necrosis; TE, thick epidermis; sTE, slight thick epidermis; tE, thin epidermis, h: hemmoarge.