An Additional risk of Lung Cancer from Recurrent Exposure to Ethyl Carbamate (EC) in BALB/C Mice

Ethyl carbamate (EC); urethane is the ester of carbamic acid. It has been found mainly as a by-product of fermented foods and beverages. EC is widely distributed in fermented foods and beverages, such as spirit, wine, beer, bread, soy sauce, and yoghurt, due to unintentional fermentation during production or storage process of fermented foods or beverages [1]. EC has been formed from substances like urea, hydrogen cyanide, citrulline, and N-carbamyl compounds during foods and beverage fermentations [2]. Earlier studies reported that unrecorded wine is rich with EC due to reaction between ethyl alcohol and hydrogen cyanide or urea wine [3]. The Joint Food and Agriculture Organization of the United Nations (FAO)/World Health Organization (WHO) Expert Committee on Food Additives (JECFA) reported that EC increase the probable health risk of carcinogenicity of certain types of alcoholic beverages for regular drinkers, especially in unrecorded alcohol [3,4]. EC exhibits carcinogenic activity on different sites such as the liver, ovary, and skin in a dose dependent manner due to its potential genotoxic activity [5]. Accordingly, IARC in 2007 upgraded its classification of EC to group 2A (probably carcinogenic to humans) [6]. There is mounting evidence that suggests carcinogenic effects of EC due to formation of DNA-reactive carcinogens, in rodent cells, is also likely to occur in humans due to significant similarities with rodents (IARC). In addition, it is still limited evidence to associate between ethyl carbamate exposures with the cancer incidence in order to attain causeeffect relationship [7]. Considering the importance of determining the risk of exposure to ethyl carbamate in fermented beverages or foods, the current study aims to investigate the potential carcinogenic effect of recurrent exposure of EC in BALB/C mice.


Introduction
Ethyl carbamate (EC); urethane is the ester of carbamic acid. It has been found mainly as a by-product of fermented foods and beverages. EC is widely distributed in fermented foods and beverages, such as spirit, wine, beer, bread, soy sauce, and yoghurt, due to unintentional fermentation during production or storage process of fermented foods or beverages [1]. EC has been formed from substances like urea, hydrogen cyanide, citrulline, and N-carbamyl compounds during foods and beverage fermentations [2]. Earlier studies reported that unrecorded wine is rich with EC due to reaction between ethyl alcohol and hydrogen cyanide or urea wine [3]. The Joint Food and Agriculture Organization of the United Nations (FAO)/World Health Organization (WHO) Expert Committee on Food Additives (JECFA) reported that EC increase the probable health risk of carcinogenicity of certain types of alcoholic beverages for regular drinkers, especially in unrecorded alcohol [3,4]. EC exhibits carcinogenic activity on different sites such as the liver, ovary, and skin in a dose dependent manner due to its potential genotoxic activity [5]. Accordingly, IARC in 2007 upgraded its classification of EC to group 2A (probably carcinogenic to humans) [6]. There is mounting evidence that suggests carcinogenic effects of EC due to formation of DNA-reactive carcinogens, in rodent cells, is also likely to occur in humans due to significant similarities with rodents (IARC). In addition, it is still limited evidence to associate between ethyl carbamate exposures with the cancer incidence in order to attain causeeffect relationship [7]. Considering the importance of determining the risk of exposure to ethyl carbamate in fermented beverages or foods, the current study aims to investigate the potential carcinogenic effect of recurrent exposure of EC in BALB/C mice.

Animals and maintenance
Male BALB/c mice (5 weeks, 22-30 g) each were purchased from Animal House Colony, Pharmacology and Chemistry Research Centre, 6 th October, Egypt. Animals were maintained on a standard lab diet (protein: 160.4; fat: 36.3; fiber: 41 g/kg of metabolisable energy =12.08 MJ) and were housed in filter-top polycarbonate cages in a room free from any source of chemical contamination, artificially illuminated (12h dark / light cycle) and thermally controlled (25 ± 1ºC) at the Animal House Lab., Pharmacology and Chemistry Research Centre. All animals received humane care in compliance with the guidelines of the Animal Care and Use Committee of the Pharmacology and Chemistry Research Centre. All the procedures described below were carried out in accordance with guidelines of ethics committee of the faculty of pharmacy, Beni-Suef University, Beni-Suef, Egypt.

Experimental design
Animals within treatment groups were assigned into three groups (10 mice/group) for 120 days as follows; Group (1), untreated control; Group (2), Animals treated with single exposure of ethyl carbamate (1.5 g/Kg/i.p); Group (3), Animals treated with repeated exposure of ethyl carbamate (1.5 g/kg/i.p) at the first day and at day 60 of the treatment period.

Mice blood and lung collection
At the end of the treatment period, animals were fasted for about 12 hours, but with free access to water ad libitum. Blood samples were collected from each animal from the tail vein under ketamine (12.5 mg/ kg) and xylazine (1.5 mg/kg) anesthesia [9]. Blood samples were left to clot and the sera were obtained using cooling centrifugation at 3,000 rpm for 15 minutes and refrigerated at -20°C until analysis. The sera were used for the determination of TNF-α, IL-1 β, LDH, AFP, CEA, CYFRA21-1, and IGF-1 according to instruction of analytical kits. After the collection of blood samples, the animals were killed by over dose anesthesia and samples of the lungs were weighed (each approximately 0.05-0.1 g) and homogenized in phosphate buffer (pH 7.4) to give 20% w/v homogenate. This homogenate was centrifuged at 1700 rpm at 4°C for 10 min and the supernatant was stored at -70°C until analysis. The supernatant of lung homogenate was used for assessment of GSH activities and MDA (malondialdehyde) levels, according to method of Beutler et al., [10] and Mihara and Uchiyama, [11], respectively. Another sample of each lung was removed and fixed in 10% formalin and embedded in paraffin wax for the histological examination [12].

Statistical analysis
All data were statistically analyzed by analysis of variance (ANOVA) using the General Linear Model Procedure of the Statistical Analysis System [13]. The significance of the differences among treatment groups was determined by Waller-Duncan k-ratio [14]. All statements of significance were based on probability of P < 0.05.

Effect of ethyl carbamate exposure on serum level of LDH, and tumor markers
The effect of exposure of ethyl carbamate (EC) demonstrated in Table 1 revealed single exposure of EC showed significant increase of LDH, CEA and AFP, while the increment of LDA, CEA and AFP was pronounced in animals treated with repeated doses of EC in comparison to single exposure or control itself.

Effect of single or recurrent exposure of EC on inflammatory cytokines
The results of the current study revealed that EC induced severe changes of serum TNF-α, and IL-1β in comparison to control group. Recurrent exposure to EC showed significant increase in serum level of TNF-α and IL-1β in comparison to animals treated with a single dose of urethane ( Table 2).

Effect of ethyl carbamate on serum level of CYFRA21-1 and IGF-1
The results of the current work showed at Table 3 that animals treated with a single dose of EC exhibit significant increase of serum level of CYFRA21-1 and IGF-1, while repeated doses of EC induce serum levels of CYFRA21-1 and IGF-1 significantly in comparison to a single dose of EC.

Effect of ethyl carbamate on lipid peroxidation and oxidative stress of lung homogenate
Animal treated with a single exposure of ethyl carbamate demonstrated a significant increase of lipid peroxidation and significant reduction of GSH activities in lung homogenate. However, recurrent administration of ethyl carbamate increases the lipid peroxidation and oxidative stress that indicated by significant increment of MDA level and reduction of GSH in comparison to single exposure of EC (Table 4).

Histological examinations
The results of the current study confirmed with histological examinations that demonstrated photomicrograph of pulmonary tissues of the control group showed no incidence of tumor nodules or microscopic lesions accompanied with normal epithelization of the bronchi, bronchioles and alveoli ( Figure 1A). Mice treated with a single dose of ethyl carbamate demonstrated marked bronchiolar hyperplasia and subepithelial edema of the epithelial lining of bronchioles. Minute   focci was characterized with neoplastic cells at the peribronchial tissues and others were scattered between the alveoli ( Figure 1B). Animals treated with repeated doses of ethyl carbamate (urethane) showed pronounced lesion accompanied with focal aggregation of neoplastic epithelial cells around the bronchioles. However, repeated exposure showed a marked increase of atypism, pleomorphism and mitotic activity and tissue necrosis around bronchi in reference to a single dose of urethane ( Figure 1C).

Discussion
Nowadays there is an increase of the interest to determine and investigate the potential risk of recurrent exposure ethyl carbamate (EC) that found in the fermented beverages and wine. Animals treated with EC showed significant increase of LDH that indicated by cellular proliferation and cancer promotion [15]. It has been well documented that LDH plays an important role in the development, invasion, and metastasis of cancerous lesion such as lung cancer via accumulation of F-FDG (Fluorodeoxyglucose) [16]. On the other hand, the data of the current work showed either animals treated with a single or repeated dose of ethyl carbamate exhibited significant increase of AFP; important diagnostic marker for hepatocellular carcinoma and germ cell. Although, the elevation of AFP in lung carcinoma is very rare, but it may be due to a metastatic effect of ethyl carbamate on hepatocytes and other sites [17]. Previous studies reported that AFP could be elevated with patient of primary lung cancer due to hepatic or testicular metastasis [18]. Carcinogenic effect of EC may be due to the formation of active metabolite; Vinyl carbamate (VC), which is potent mutagen and promote formation of electrophilic species that interact with DNA to form the 2-oxoethyl adducts as vinyl chloride [19]. Animals treated with urethane showed significant increase of CEA and the increment was pronounced in the group treated with repeated doses of EC (urethane) that indicated promotion of cellular proliferation of lung [20]. The results of the current study revealed that animals treated with urethane showed significant increase of IGF-1 and the increment was enhanced in the animals treated with recurrent exposure. Previous reports demonstrated that urethane induced pulmonary adenocarcinoma through up-regulation of NF-кB that stimulate IGF-1 and inhibit other tumour suppressor genes [21]. Data of the present work of animals treated with EC showed significant increase of CYFRA21-1 that indicated the carcinogenic effect of ethyl carbamate [22]. CYFRA21-1 is the epithelial cell markers and very sensitive and specific markers for non-small cell lung cancer [23]. Recurrent exposure to ethyl carbamate induced sever changes of lipid peroxidation and oxidative stress that indicated by significant increment of MDA (malondialdehyde) and reduction of GSH in lung homogenate in comparison to control group. This could be attributed to increase of intrinsic ROS generated in the mitochondria through formation of electrophilic species with permanent mitochondrial dysfunction of lung cells [24]. Mitochondrial dysfunction and oxidative stress of repeated exposure to EC may be over stimulation of IFN-γ-producing  cells, CD11b + Gr-1 + phenotype [25] as well as overexpression NF-кB, COX-2, STAT3, IL-6 and cyclin D1 [26]. The results of the current study showed either single or repeated exposure to ethyl carbamate resulted in a significant increase of inflammatory cytokines; TNF-α, IL-1β in comparison to control group. The results were in agreement with previous work that reported EC induce over expression of key molecules such as NF-κB, Stat3, pStat3; IL-1β. They play a major role in pulmonary inflammation and adenocarcinoma [27]. It has been well documented that the molecular mechanism of ethyl carbamateinduced lung cancer is based on integration of TNF signals with NF-кB that accelerate tumour progression [28]. Histological studies confirmed the biochemical results of the current work. These results are in accordance with previous study by Nakahara et al. [29]. Microscopic examination showed that recurrent exposure of EC showed aggregation of epithelial cell due to significant increment of ROS production and ERK activation in macrophages that leads to enormous production of lung epithelial cells. The results of the current study demonstrate that ethyl carbamate is an additional risk of lung carcinoma and other types of cancer in fermented beverage and should encourage implicating a policy for prevention and counteracting unrecorded and contaminated beverages.