Toxicological evaluation of some Malaysian locally processed raw food products

Toxicological evaluation of some Malaysian locally processed raw food products R. Sharif b, A.R. Ghazali b a,* , N.F. Rajab a, H. Haron b, F. Osman c a Biomedical Science Department, Faculty of Allied Heath Sciences, Universiti Kebangsaan Malaysia, 50300 Kuala Lumpur, Malaysia Nutrition and Dietetics Department, Faculty of Allied Health Sciences, Universiti Kebangsaan Malaysia, 50300 Kuala Lumpu, Malaysia c Ministry of Health, Department of Food Quality Control, National Health Laboratory, Sungai Buloh, Selangor, Malaysia Abstract Malaysian locally processed raw food products are widely used as main ingredients in local cooking. Previous studies showed that these food products have a positive correlation with the incidence of cancer. The cytotoxicity effect was evaluated using MTT assay (3-(4,5-dimetil-2-thiazolil)-2,5-diphenyl-2H-tetrazolium bromide) against Chang liver cells at 2000 lg/ml following 72 h incubation. Findings showed all methanol extracts caused a tremendous drop in the percentage of cell viability at 2000 lg/ml (shrimp paste – 41.69 ± 3.36%, salted fish – 37.2 ± 1.06%, dried shrimp – 40.32 ± 1.8%, p < 0.05). To detect DNA damage in a single cell, alkaline Comet Assay was used. None of the extracts caused DNA damage to the Chang liver cells at 62.5 lg/ml following 24 h incubation, as compared to the positive control, hydrogen peroxide (tail moment – 9.50 ± 1.50; tail intensity – 30.50 ± 2.50). Proximate analysis which was used for the evaluation of macronutrients in food showed that shrimp paste did not comply with the protein requirement (<25%) as in Food Act 1983. Salt was found in every sample with the highest percentage being detected in shrimp paste which exceeded 20%. Following heavy metal analysis (arsenic, cadmium, lead and mercury), arsenic was found in every sample with dried shrimps showing the highest value as compared to the other samples (6.16 mg/kg). In conclusion, several food extracts showed cytotoxic effect but did not cause DNA damage against Chang liver cells. Salt was found as the main additive and arsenic was present in every sample, which could be the probable cause of the toxicity effects observed. Keywords: Malaysian local raw food products; Shrimp paste (belacan); Salted fish; Dried shrimp; Chang liver cells; MTT assay; Alkaline Comet assay 1. Introduction Diet plays a major role in cancer etiology and prevention (AICR, 1997). Previous epidemiological studies, supported by preclinical data from animal and in vitro experiments and by clinical findings, have contributed immensely in providing insights into links between diet and cancer prevention and to the development of diet and cancer hypotheses. The first study on diet and cancer was carried out by Doll and Peto (1981). They attempted to quantify the environmental contributions of a variety of factors including * Corresponding author. Tel.: +603 40405618; fax: +603 26929032. E-mail address: rohi@medic.ukm.my (A.R. Ghazali). diet, alcohol, tobacco, occupation and radiation. Following this, studies on the effect of diet on cancer were conducted. Current evidence suggests that genotoxic carcinogens for many cancers stem from the traditional intake of fried and broiled foods such as meat (Sugimura, 1996) containing a new class of powerful mutagens, heterocyclic amines, which are carcinogenic in animal models (Weisburger, 1997). Carcinogens in the diet that trigger the initial stage of cancer include nitrosamines (in smoked and cured products), heavy metals, additives and preservatives (Reddy et al., 2003). Various factors, including salted and pickled food contributed to the development of hypotheses which suggested a relationship between diet and cancer. A strong and consistent correlation between the intake of salt and salted food and the incidence of stomach cancer and other pre-cancerous lesions has been reported (Yamaguchi and Kakizoe, 2001; Montes et al., 1985). In Japan, a novel type of chemical, 2-chloro-4-methylthiobutanoate was found in Japanese salted fish that acted as a powerful direct-acting mutagen (Chen et al., 1996). Interestingly, this chemical induces DNA repair in gastric mucosa, similar to the effect of the classic gastric carcinogen N-methyl-N-nitro-N-methylnitrosoguanidine (Furihata et al., 1996). Besides gastric cancer, salted food was also found to be linked with a risk of other cancers, specifically nasopharyngeal carcinoma (NPC). Numerous epidemiological studies have been done to identify the major risk of NPC. In China, the consumption of salted fish and shrimp paste was shown to be a significant risk factor for NPC (Yuan et al., 2000). In Malaysia, salted food was found to exhibit strong positive associations with the incidence of NPC (Armstrong et al., 1998). A previous study had shown that various foods processed with nitrite from Japan and South East Asia had direct mutagenic effect (Wakabayashi et al., 1985). In addition, animal studies reported that fried salted fish could also induced maximum chromatid damage when fed to rats (Taj and Nagarajan, 1994). The presence of nitrosamines, known to be potent carcinogens has been detected in salted fish consumed in Southern China (Fong and Chan, 1977; Huang et al., 1977; Huang et al., 1978). Experimental investigations also indicated that rats fed with Chinese salted fish developed carcinoma of the nasal and paranasal cavities (Huang et al., 1978; Yu et al., 1989; Zheng et al., 1994). Furthermore, N-nitrosodimethylamine and 7-methylguanine DNA adducts were found in the tissue of rats which had been fed with Chinese salted fish (Widlak et al., 1995). Due to a large body of evidence to suggest that salted food is a cancer risk, we tested some Malaysian raw food products to assess the level cancer risk. The products we tested were salted and fermented food, i.e. shrimp paste, dried shrimp and salted fish. These are widely consumed and commonly used in everyday cooking. Earlier studies had shown that these food items contained saturated salts which could be linked to pathogenesis of cancer (Ghazali et al., 2005). The purpose of the present work is to investigate the cytotoxicity and genotoxicity effects of several locally processed food products in vitro and the level of contaminants, including additives, preservatives and heavy metals from each of the samples that may have implications on human health. Proximate analysis was conducted to determine the macronutrients in each sample. 2. Materials and methods 2.1. Food samples All the food samples, i.e. salted fish, shrimp paste and dried shrimps were purchased from Malacca which is a main production and distribution centre for these foods. Half of the food was used for food analysis and the other half for extraction. 2.2. Extraction of food samples The ground powder (200 g) of each of the food samples was soaked in 500 ml methanol (Chemical Industries, Malaysia) for two days. The mixture was then filtered and the solvent evaporated using a rotary evaporator (Buchi Rotavapor R-114, Switzerland) at 50 C. The resulting pellet was then freeze-dried (Heto LyoLab 3000, Denmark) and the powder extract was kept at 4 C in an air-tight jar prior to the bioassays. For aqueous extraction, the ground powder (200 g) of the food was soaked in 500 ml of distilled water for 24 h and stored at 4 C in the dark to prevent microbial activity. The mixture was then filtered freeze-dried and the dry extract kept at 4 C in an air-tight jar prior to the bioassays. 2.3. Reagents and cells Human Chang liver cells were obtained from ATCC (Rockville, MD, USA) and cultured as described previously (ATCC Catalogue Details No CCL 13, 2003). Cells were grown as monolayers in a T-25 cm2 culture flask. The medium was supplemented with 2.0 g/l sodium bicarbonate, antibiotics (100 U of penicillin/ml, 100 lg of streptomycin/ml) and 10% fetal bovine serum. The cell culture medium and their supplements were purchased from Life Technologies, Gibco BRL Products (Rockville, MD). The cell cultures were maintained in a humidified atmosphere of 5% CO2 at 37 C and were harvested when they reached 80% confluency, i.e., in their exponential growth phase. For bioassay activity, methanol and aqueous extracts of each sample were dissolved in 5% dimethyl Sulphoxide (DMSO) (Chemical Industries, Malaysia) and media RPMI-1640 (Flowlab, USA) to a final concentration of 10 mg/ml. These solutions were then filtered using sterile 0.45 lm syringe filter. Hydrogen peroxide at 100 lM was used as the positive control for MTT assay and 0.1 lM for alkaline Comet Assay. 2.4. The MTT cytotoxicity assay The viability of the Chang liver cells was used to determine the cytotoxicity effect of each of the food samples as described previously (Mosmann, 1983). The cell monolayers in exponential growth were harvested and 5 · 103 cells in 100 ll were placed into each well of the 96-well plates (NunclonTM, VWR International Inc., MD). The plates were incubated for 24 h at 37 C in 5% CO2. The medium was discarded and 200 ll of the test extracts for each sample in different concentrations were loaded into the 96-well plates. After 72 h incubation, 20 ll of the MTT solution was added to each well and reincubated for 4 h at 37 C before the medium was discarded and 100 ll of DMSO added to dissolve the formazan crystals. The plate was shaken for 30 min to dissolve the crystals formed and the absorbance was measured at 570 nm with a microplate reader. Assays with each concentration were repeated three times. The MTT (3-(4,5-dimethylthiazol-2-yl)-2-5-diphenyl tetrazolium bromide) (Sigma Chemical Co., St. Louis, MO) used was dissolved in phosphate buffer saline (PBS) solution at concentration of 5 mg/ml and filtered through a 0.22 lm filter to sterilize and remove insoluble residues. 2.5. Alkaline Comet Assay (Singh et al., 1988) Chang liver cells were used in this study as described previously. A total of 8 · 104 cells in 2 ml were placed into each well of a 6-well plate (NunclonTM, VWR International Inc., MD, USA) and incubated for 24 h at 37 C in 5% CO2. In an assay, the medium was discarded and test extracts at a concentration of 62.5 lg/ml (IC25) were added for 24 h. A negative control was used, consisting of the medium without any extract. After 24 h incubation, cells were washed with PBS and trypsinized to detach the cells. The cells were transferred to eppendorf tubes and centrifuged at 2500 rpm for 5 min. The pellet was resuspended in PBS and recentrifuged. Frosted slides were prepared with a layer of normal melting agarose (Sigma Chemical Co., St. Louis, MO, USA). Cells were then suspended in low-melting point agarose (Sigma Chemical Co., St. Louis, MO, USA) maintained at 37 C and placed on the slides coated with normal melting agarose. After the agarose gel had solidified, the slides were placed for at least 1 h in a lysing solution consisting of high salts and detergents (100 mM ethylenediaminetetraacetic acid [EDTA], 2.5 M sodium chloride, 10 mM Trizma base, adjusted to pH 10) with 1% Triton X-100 added just prior to use. The slides were then incubated in alkaline (pH > 13) electrophoresis buffer (1 mM EDTA and 300 mM sodium hydroxide) for 20 min to produce single stranded DNA. After unwinding, the single stranded DNA in the gel was electrophoresed under alkaline conditions at 25 V and 300 mA for 20 min to produce comets. The alkaline buffer used during electrophoresis was the same as the unwinding buffer. The alkali in the gels then was neutralized by rinsing the slides thrice with Trizma buffer at pH 7.5 for 5 min each time. Finally, slides were stained using ethidium bromide. The slides were analyzed using a fluorescent microscope (Leitz Laborlux Epifluoresence Microscope, Germany) equipped with 515 barrier filter and 560 emission filter. 2.6. Determination of the levels of Food Additives and Heavy Metals (AOAC, 1995) All the analyses were conducted at the National Health Laboratory in Sungai Buloh, Selangor. The determination of the food additives and heavy metals was conducted according to the standard Food Quality Control Department, National Health Laboratory (FQCD) method. Benzoic acid and sorbic acid in foods were determined using liquid chromatography (internal standard). Water soluble synthetic food colouring in the foods was determined using paper chromatography and UV/ VIS spectrophotometer. The Volhard method was used to measure sodium chloride in salted food. The determination of mercury was conducted by atomic absorption spectrophotometry (AAS)-FIAS. Other heavy metals (lead, arsenic and cadmium) were determined using inductively coupled plasma optical emission spectroscopy (ICP-OES). 2.7. Proximate foods analysis Moisture, total ash, crude protein, fat and total carbohydrate were determined using standard method (AOAC, 1995). Moisture was determined by drying 5 g samples in a vacuum oven at 105 C for 24 h to a constant weight. The nitrogen content was estimated by micro-kjeldhal techniques using Tecator System (Tecator, United Kingdom). Ash was determined by ignition at 550 C in an electric furnace (Carbolyte, United Kingdom). Fat was determined using Soxtec system (Soxtec, United Kingdom) and total carbohydrate was calculated using difference of the value of each nutrients. 2.8. Statistical analysis ANOVA was used to measure significant differences between the means. 3. Results At 2000 lg/ml, all methanol extracts showed a tremendous drop in the percentage of cell viability (shrimp paste – 41.69 ± 3.36%, salted fish – 37.2 ± 1.06%, dried shrimp – 40.32 ± 1.8%, significant, p < 0.05). Aqueous dried shrimp extract showed the least reduction in cell viability where at maximum concentrations (2000 lg/ml), 93.61 ± 4.81% cell viability was reported compared to the other extracts (aqueous salted fish extract – 75.70 ± 4.85%; aqueous shrimp paste extract – 73.85 ± 4.22%) (see Fig. 1). In this assay, scores were given according to the DNA damage of the cell using software Comet Assay Analysis System, Kinetics, USA. Tail moment and tail intensity were used to determine the DNA damage. Tail moment is defined by the product of the distance between the head and the tail by the proportion of DNA in the tail, was used to evaluate the extent of DNA migration (Olive et al., 1990) while tail intensity refers to the percentage of DNA at the tail of the comet (see Figs. 2 and 3). None of the extracts showed any severe DNA damage compared to the negative control. Fig. 4 shows tail moment and Fig. 5 shows tail intensity for each of the samples used in this study. Aqueous shrimp paste extract and salted fish exhibited higher DNA damage (TM – 0.99 ± 0.33; TI – 6.18 ± 2.51, TM – 0.84 ± 0.48; TI – 5.30 ± 2.18, respectively), than the other extracts. On the other hand, both methanol shrimp paste extract (TM – 0.83 ± 0.28; TI – 4.51 ± 0.74) and salted fish extract (TM – 0.75 ± 0.27; TI – 4.55 ± 1.62) showed slightly high value for tail moment and tail intensity as compared to negative control (TM – 0.29 ± 0.05; TI – 2.50 ± 0.29). Dried shrimp extract caused the least damage to the DNA of the cell (methanol extract – TM – 0.322 ± 0.13; TI – 3.56 ± 1.09, aqueous extract – TM – 0.35 ± 0.11; TI – 3.49 ± 1.21). The captured image of cells in Figs. 5 and 6 show that there was no DNA damage in the cells in the negative control and the methanol extracts of dried shrimps from Batang Tiga. Cells were intact and the comet tail was not observed in the slides. However, the image in Fig. 6 shows cells with DNA damage following treatment of hydrogen peroxide (positive control) at 0.1 lM for 30 min at 4 C. The comet tail was observed in the slide and DNA of the cells lysed (Fig. 6). In the food additives analysis, the results showed no evidence of the presence of sorbic acid, benzoic acid or synthetic colouring. Shrimp paste and salted fish contained the highest percentage of salt (sodium chloride) with 20% followed by dried shrimp with only 4% (see Table 2). However, total arsenic was found in each food item. Other heavy metals such as lead, cadmium and mercury were not present in all the samples. The levels of total arsenic in each food item was much higher compared to the level permitted by the Malaysian Food Act 1983 (1 mg/kg). Shrimp pastes contained the highest level of total arsenic with 6.16 mg/kg followed by dried shrimps with 4.03 mg/ kg and salted fish which contained the least (1.89 mg/kg) (see Table 1). Proximate analysis was conducted to evaluate the macronutrients of each food analysis. From the graph (Fig. 7), the moisture content differed between samples in with the moisture content for shrimp pastes being higher than for salted fish and dried shrimps (36.70 ± 2.40%, 29.20 ± 1.98% and 29.00 ± 0.57%). The highest level of total ash was found in shrimp paste with 34.26 ± 2.79% followed by salted fish with 26.63 ± 1.10% and dried shrimps with 12.43 ± 0.15%, respectively. The highest value for crude protein was presented by the dried shrimps with 46.50 ± 0.99% whereas shrimp paste contained the lowest percentage of crude protein with 23.43 ± 0.34%. For total carbohydrate and fat, the content of all the SFM SFA SPM SPA DSM DSA Percentage of cells viability (%) 110 100 90 80 70 60 50 40 30 20 10 0 0 500 1000 1500 2000 Concentrations (µg/m l) Fig. 1. Percentage cell viability (%) following incubation with food extracts at different concentrations. Each point represents the mean ± SEM of three different independent experiments. Abbr: SFM – salted fish methanol, SFA – salted fish aqueous, SPM – shrimp paste methanol, SPA – shrimp paste aqueous, DSM – dried shrimp methanol, DSA – dried shrimp aqueous. 12 Arbituary Unit 10 8 6 4 2 0 Negative Positive SFM SFA SPM Samples SPA DSM DSA Fig. 2. Bar chart of tail moment based on the Comet Assay scoring of its DNA damage induced by the Malaysian food items (mean ± SD) (n = 50 cells, experiments = 3). 35 % of DNA in tail 30 25 20 15 10 5 0 Negative Positive SFM SFA SPM Samples SPA DSM DSA Fig. 3. Bar chart of tail intensity based on the Comet Assay scoring of its DNA damage induced by the Malaysian food items (mean ± SD) (n = 50 cells, experiments = 3). Fig. 4. Chang liver cells processed in the comet assay following negative treatment (25· magnification). Fig. 6. Chang liver cells processed in the comet assay following treatment with 0.1 nM Hydrogen Peroxide for 30 minutes at 4 C (25· magnification). Fig. 5. Chang liver cells processed in the comet assay following treatment with methanol extracts from shrimp pastes for 24 h (25· magnification). effect caused by the food extracts. High salt content can cause the death of the cells in mechanisms such as osmosis or alteration to the homeostasis of the cells (Cohen and Roe, 1997). However, based on the classification of cytotoxicity by Abbas and friends (1984), at 2000 lg/ml, both methanol extracts (dried shrimp and shrimp paste) showed weak cytotoxic effect. All the other extracts showed no effect of cytotoxicity at all. In this present study, no direct DNA damage effect was seen. The previous report found extracts of salted foods to be mutagenic and clastogenic in an in vitro system but not causing any direct DNA damage (IARC, 1993a, 1993b). High levels of arsenic were found in all samples. This could be due to the ability of microorganisms in the environment to convert arsenic to dimethylarsenate, which then accumulates in fish (Hodgson and Levi, 2000). Besides, arsenic may also be a contaminant during processing and may also be from ground water that has been used in the processing of the product. The common regulation refers to total arsenic on the assumption that it would be mainly inorganic. The maximum permissible concentration of arsenic in food is currently 1.0 mg/kg. The daily intake of arsenic by humans reflects the quantity of seafood in the diet in which arsenic occurs mainly in the organic form. Among marine animals, arsenic is found to be accumulative to levels from 0.005 to 0.3 mg/kg in coelenterates, some molluscs and crustaceans (Bowen, 1966). Some shellfish may contain over 100 lg/g of arsenic. The average arsenic content in freshwater fish is of 0.54 lg/g total wet weight, samples showed a minimal percentage and indicated that all the food samples were low in fat and carbohydrate. 4. Discussions Based on the MTT graph, all extracts showed a reduction in the percentage of cell viability which could be due to the high amount of salt in the samples. With reference to the Malaysian Food Act. (2004), the amount of salt is higher than the standard value which is 15% for all processed shrimp paste. The difference in the amount could be one of the reasons for the difference in the cytotoxic Table 1 Detection of heavy metals on malaysian foods samples Samples Cadmium (mg/kg) Lead (mg/kg) Arsenic (mg/kg) Mercury (mg/kg) Salted fish Shrimp paste Dried shrimp Not detectable Not detectable Not detectable Not detectable Not detectable Not detectable 1.89 6.16 4.03 Not detectable Not detectable Not detectable Salted Fishes Shrimp Pastes total ash crude protein Dried Shrimps 50 45 Percentage (%) 40 35 30 25 20 15 10 5 0 water crude fat total carbohydrate Components Fig. 7. Histogram of percentage of every nutrient components for each sample (mean ± SEM), n = 3. Table 2 Detection of food additives and preservatives on malaysian foods samples Samples from Malacca Benzoic acid Sorbic acid Synthetic colourant Salt content (%) Salted fish Shrimp paste Dried shrimp Not detectable Not detectable Not detectable Not detectable Not detectable Not detectable Not detectable Not detectable Not detectable 20 20 4 but some values could reach as high as 77.0 lg/g in the liver oil of freshwater bass (Whitacre and Pearse, 1972). The majority of arsenic in marine fish is in the form of arsenobetaine (Phillips, 1990) which is excreted unaltered by the metabolism of mammalian consumers and is therefore of low toxicological significance (Kaise et al., 1985). In addition, arsenic was found to promote genetic damage by inhibiting DNA repair (Bencko et al., 1988; Astolfi et al., 1981). Besides, another point of genotoxic effect of arsenic is found when enzymes such as superoxide dismutase and catalase that scavenge for oxygen free radicals seem to provide protection against arsenic-induced DNA damage (Mandal and Suzuki, 2002). Arsenic may cause DNA damage by inhibiting DNA repair mechanisms where it can bind strongly to dithiols and sulfhydril group. Such protein binding can induce inhibited DNA repair, mutation in key genetic sites, or increased cell proliferation, which could then lead to subsequent mutation via inhibited DNA repair (Mandal and Suzuki, 2002). The Malaysian Food Act 1983 (Act 1981) and Regulations, states that shrimp paste should not contain less than 15% of salt and 25% of protein and should not contain more than 40% of water and 35% of ash. As compared to the standard value in the Food Act, our shrimp paste sample did not comply with the protein standard. 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