Genotoxic Potential of Shrimp Pastes ( Belacan ) Extracts Using Umu Test

This study was conducted to observe the genotoxic effects of aqueous, methanol, hexane and dichloromethane extracts of “belacan” (shrimp paste) taken from three local districts in Melaka, Malaysia (Kelemak, Batang Tiga & Pantai Puteri). The umu test which was used as the screening test was conducted with and without the presence of metabolic activation system. Without the presence of metabolic activation system, aqueous extracts from Kelemak showed mutagenicity activity at 5 mg/ml with IR (Induction Rate) = 1.52 ± 0.57 and the methanol extracts showed mutagenic activities at 0.625 mg/ml and 5 mg/ml, which the IR was the highest at 5 mg/ml (2.08 ± 0.09). On the other hand, samples from Batang Tiga, Melaka showed mutagenic effects at all five concentrations for the dichloromethane extract, with IR = 2.09 ± 0.64 as the highest value at 1.25 mg/ml. Methanol extracts also showed positive results at 1.25 mg/ml and 2.5 mg/ml with IR = 1.70 ± 0.33 and IR = 2.12 ± 0.51 respectively, and aqueous extract at 0.625 mg/ml with IR = 1.54 ± 0.48 and 5 mg/ml with IR = 1.74 ± 0.50. There was a significant difference of the mean values of IR between the four different types of “belacan” extracts from Batang Tiga (p < 0.05). All four “belacan” extracts from Pantai Puteri, Melaka did not show any mutagenic effect. With the presence of metabolic activation system, there was no mutagenic effect observed in all four extracts from the three districts. Further study to analyze the contents in the food samples should be done in the future to determine the possible contents in the food samples that might be responsible for the mutagenic activities.


Introduction
Various factors such as the environment, diet, lifestyle, health status and genetics can influence human health.Diet along with alcohol consumption and smoking habit are the most significant factors of different kinds of diseases [1].In addition, various evidences from ecological and experimental experiments showed that diet has a role in the etiology of cancers.
"Belacan" (shrimp paste) is one of the most consumed local raw food products daily in Malaysia [2].It is made from wet shrimps fermented with 10% -15% w/w of salt.According to Arshad et al. [3] "belacan" is enlisted as one of the seven foods that associated with cancer which are anchovies, dried fish, salted fish, shrimp paste, pickled fruits, salted egg and pickled vegetable.Salted fish has been proven to be carcinogenic to human [4].Salted fish is found to contain high content of nitrosamine as well as a few other chemicals [5].
Toxicological evaluations on several raw food products had been done by Ghazali et al. [6] and "belacan" at 62.5 µg/ml caused the highest DNA damage on Chang liver cells, followed by salted fish, anchovies and dried shrimps.Studies on animals had shown that 10% of salt in diet causes the induction of mucosal damage such as cell degeneration, and further destroy the mucosal membrane lining the rat gaster [7].Further studies of chemical analysis and animal experiments have to be conducted in order to examine into details about the carcinogenic potential of food products preserved by salting treatment.Preservation process using salt and drying can also lead to the formation of N-nitroso compounds.Nitrite in salts will form a reaction with amides and amines in meats or proteins which produces N-nitroso compounds [8].The objective of this study was to evaluate genotoxicity potential of shrimp paste from Malaysia using umu test.

Sampling
Food samples were obtained by simple random sampling method in Malacca, which is one of the main producers of "belacan" in Malaysia.Samples are selected from three local districts which are Kelemak, Pantai Puteri and Batang Tiga.Extraction using dichloromethane, methanol, hexane and aqueous were done for sample preparation.Dichloromethane and hexane are non-polar solvent, methonal is a polar protic solvent and water is the most polar solvent [9].

Umu Test
The method for analysis was umu test whereby the Salmonella typhimurium TA1535/pSK1002 strain was obtained from Professor Dr.I Aoyama from Okamaya University, Japan.Salmonella typhimurium TA1535/ pSK1002 was used in umu test.Salmonella typhimurium TA1535/pSK1002 was derived from its precursor strain which was Salmonella typhimurium TA98, a mutant bacteria.This strain carries plasmid pSK1002 harboring a fusion gene umuC'-lacZ.Plasmid pSK1002 also coded the genes for ampicilin resistance.The genetic markers of this strain were hisG, uvrB, rfa, umu-ClacZ and amp.The umu test had been standardized by the German Institute for Standardization (DIN 38 415 T3) and also by ISO (ISO/DIS 13829, 2000) to detect genotoxins in various compounds including environmental and food samples.
The samples were tested with and without the S9 metabolic system [10].S9 mix is a liver enzymes fraction pooled from male Sprague-Dawly (Sigma-Aldrich) and used for activation of promutagen substances to mutagen.Protocols for umu test consisted of genetic analysis, growth curve, and preparation of bacteria stock and umu test.For the umu test, IR (Induction Rate) = 1.5 is reported as a positive result [11][12][13], 4-nitroquinoline-1oxide (4-NQO) (for without S9 metabolic system) and 2-aminoanthracene (2-AA) (with presence of S9 metabolic system) were also used as the positive controls.
There are a few factors that might cause the observed positive results.According to previous studies by Sharif et al. [14], salt content analysis on "belacan" samples obtained from Malacca was 20%, whereby Sumino et al. [15] found that "belacan" processed in Malaysia contain approximately 24.1% salt.According to Malaysian Food Act 1983, the standard of salt content for "belacan" should be around 15%. High concentration of salt can lead to chromosome breakage [16], cell death and also can induce DNA damage [6].According to Cohen & Roe [16], sometimes, the usage of salts contaminated with nitrite and nitrates can also lead to nitrosamines contamination in foods.It is also suggested that the presences of N-nitroso compounds is one of the factors that can also lead to risk of cancer.
The extracts of "belacan" only showed mutagenic effects without metabolic activation.S9 metabolic active-  tion system acts as an enzyme for the metabolism of xenobiotic compounds whereby toxic compounds will undergo biotransformation into less toxic compounds [17].Therefore, mutagens that are present in the samples could have been transformed into less toxic compounds and do not show any mutagenic activity.

Conclusion
The study showed that the "belacan" samples were found to induce genetic damage to Salmonella thyphimurium without the presence of metabolic activation.However, samples tested with umu test with the presence of metabolic activation did not show any positive result.

Table 1 . IR of "belacan" extracts from Kelemak, Melaka without metabolic activation.
*Significantly difference as compared to control.

Table 3 . IR of "belacan" extracts from Batang Tiga, Melaka without metabolic activation.
*Significantly difference as compared to control.