TTX and Genotoxicity of Diodon Hystrix Organs

TTX and Genotoxicity of Diodon Hystrix Organs Identification of TTX and Genotoxicity of Diodon hystrix Organs Adwaid Manu K, Vignesh M., Riven Chocalingum Abstract Tetrodotoxin is alkaloid based aquatic toxins. These toxins are one of the most potent non-proteinaceous toxins as well as the best-known marine natural toxins. Diodon hystrix (porcupine fish) were collected from Chennai costal region and dissected under sterile conditions to obtain: liver, skin, gonads, intestine, eyes and kidney. 20g of each organ was macerated in 200ml of Methanol:Acetic Acid [99:1]. The filtrate is then condensed in Rota-Vaccum evaporator to obtain crude extract. The focus of this study is to confirm the presence of TTX (Tetrodotoxin) in six different organs of Diodon hystrix. Analytical techniques used were GC-MS and UV spectroscopy. Also, genotoxicity of the crude extract were analysed using human leukocyte culture and SCE assay using onion root tips. The results suggest the presence of TTX in major skin, liver and intestine and that, the organ extract does not have any genotoxic effect but is capable of increasing the sister chromatid exchange. Key Words: TTX, Diodon hystrix, genotoxicity, root tip assay. Introduction Tetrodotoxin (TTX) is a very powerful alkaloid neurotoxin that is non-proteinacious in nature. TTX can withstand very high temperature and is water soluble but is affected by extreme pH conditions, i.e., above 8.5 and below 3.0 [1, 2, 3, 4, 5]. These properties make it a dangerous toxin capable to interact best with its environment [1, 2, 5]. It is found in both aquatic as well as terrestrial organisms and studies have proven that it is synthesized by symbiotic microorganisms, bacteria precisely, present in the gut, initially acquired through the food chain or found on the skin of the animals but its biosynthesis pathway is still unknown [ 1, 2, 5, 6, 7, 8]. TTX acts as an ion pore blocker, binding to site 1 sodium channel receptor of the axon membrane thus inhibiting the influx of sodium ions and therefore leading to the blockage of action potentials [1, 2, 3, 4, 5, 6, 7, 8, 9]. TTX is ten thousand times poisonous than cyanide and one of the most fatal poisons on Earth. The LD50 is approximately 0.2ÃŽ ¼g when injected in mice [2, 5]. On the other hand, along with the lethal characteristics, clinical trials and research studies have demonstrated that TTX has remarkable therapeutic properties as an analgesic in cancer treatment process [2]. Puffer fish belonging to the order Tetraodontiformes, had been identified to be the cause of many mortalities due to food poisoning as a result of TTX intoxication. In many countries such as Japan and China, puffer fish is regarded as a food delicacy provided that it is prepared by a licensed and well experienced chef but some cases of poisoning still prevail [1, 3, 4, 5, 6, 7, 8, 9, 10]. It has been reported that only a very low dose of TTX in blood is adequate for an immediate impact on the host [5]. Studies have concluded that the most toxic organs of the puffer fish are the liver followed by the intestine and then the skin and ovary. In addition to that, TTX is also found in low concentration in other organs such as the eyes and muscles [3, 5, 8, 10]. The study is focused on Diodon hystrix which is a type of puffer fish belonging to the class Diodontidae and it is also known as Porcupinefish because of the sharp needle-like structures covering its entire body as a defense mechanism against predators. Presence of TTX has been reported in Diodon hystrix around the world [2, 4, 5] but studies on this animal from the sea of the eastern coast of India that is the Bay of Bengal is yet to be reported. The aim of this research is to identify TTX in the crude extract from Diodon hystrix collected from Chennai Coastal line and to investigate the Genotoxicity of the crude extract from respective organs using human leukocyte culture and onion root tips. Materials and methods Sample collection The puffer fish was collected from the coastal lines of marina beach, Chennai in early July 2014. The identification of the puffer fish was done by visual comparison with an online fish database www.fishbase.org. The database parameters were set accordingly to sample collection site and the possible species available in Bay-of-Bengal region with the matching morphology were only two types of Diodon sp.. Out of which Diodon hystrix had the closest match, based on the skin coloration pattern. Organ separation and extraction process The collected puffer fish were dissected and visceral organs like liver, intestine, kidney, eye, and skin were removed and organs were weighed. The isolation for the tetrodotoxin[3] include from the samples 10 grams of organs were taken and Then suspended in 100ml of three volume of 1% acetic acid in methanol without damaging the tissues then the whole materials were in the Refrigerator for 24 hours at a sterile condition, as an incubation period In the next step the tissue were macerated in a mortar and pestle gently, if the tissues get dried up add required volume of the chilled ethanol if needed. Then the slurry were filtered by using whatman no. 1 filter paper. Then the filtrate solutions were centrifuged at 12000 rpm for 10 minutes at 4 degree Celsius. Then the supernatant were separated and lastly the samples were concentrated by using lyophilisation to obtain crude extracts for our purpose of study Dragendorff’s test To identify the presence of alkaloids [10] to 2mg of crude extracts 5ml of distilled water were added and then 2M hydrochloric acid was added until an acid reaction occurs. To this 1 ml of Dragendorff’s reagent was added. Formation of orange or orange red precipitate indicates the presence of alkaloids GasChromatography-Mass Spectrometry Gas chromatography (GC) and mass spectrometry (MS)[8][11][12]forms an effective combination for Chemical analysis. GC-MS analysis were an indirect method to detect TTX in a crude extract, which was difficult to purify in other advanced analysis methods. In this method, we dissolved TTX and its derivatives in 2 ml of 3 M NaOH and heated in a boiling water bath for 30 min. After cooling to room temperature, the alkaline solution of decomposed compounds was adjusted to pH 4.0 with 1N HCl and the resulting mixture was chromatographed on a Sep- Pak C18 cartridge (Waters). After washing with H2O first and then 10% MeOH, 100% MeOH fraction were collected and evaporated to dryness in vacuo. To the resulting residue, a mixture of N, O-bis acetamide, trimethylchlorosilane and pyridine (2: 1: 1) was added to generate trimethylsilyl (TMS) ‘‘C9-base’’ compounds. The derivatives were then placed in a Hewlett Packard gas chromatograph (HP-5890-II) equipped with a mass spectrometer (AutoSpec, Micromass Inc., UK). A column (φ 0.25 mm Ãâ€" 250 cm) of UB-5 was used, and the column temperature is increased from 180 to 250 °C at the rate of 5 or 8 °C/min . The flow rate of inlet helium carrier gas were maintained at 20 ml/min. The ionizing voltage is generally maintained at 70 eV with the ion source temperature at 200 °C. Scanning was performed in the mass range of m/z 40–600 at 3s intervals. The total ion chromatogram (TIC) and the fragment ion chromatogram (FIC) were selectively monitored. Ultraviolet (UV) spectroscopy In UV spectroscopy, TTX was generally determined by irradiating a crude toxin with UV light [11][12]. A small amount of samples were dissolved in 2 ml of 2 M NaOH and heated in a boiling water bath for 45 min. After cooling to room temperature, samples were examined in UV spectrum and results were observed in the range 270nm to 280nm. Genotoxicity Human Leukocyte Culture Chromosome preparations were obtained from PHA-stimulated peripheral blood lymphocytes[14][15]. To the fresh tubes 5ml of Hikaryo XL RPMI ready-mix media and 0.5ml of heparinized Blood (50drops) were added and the contents were mixed gently by shaking. Then Incubated for 72 hours in standing position in an incubator. At the end of 48th hour of incubation, the culture was treated with TTX (0.5ug/ml) (10ul/ 5ml of culture) and again kept it in incubator for another 24 hours. At the end of 24th hour incubation, the culture was thoroughly washed by centrifuging the content at 1500rpm for 5 minutes, discard the supernatant and add 5ml of RPMI 1640 medium. To the content 60 microliter of colchicine was added and tubes were kept for 20 minutes incubation in incubator at 37oC and the content was centrifuged at 1500 rpm for 10 minutes after incubation. The supernatant was removed and 6ml of pre-warmed 0.075M hypotonic solution was added. The content was mixed using a Pasteur pipette and incub ated at 37 oC in incubator for 6 minutes. After incubation the content tube was centrifuged at 2000 rpm for 5 minutes. The supernatant was discarded and 6ml of Carnoy’s fixative was added and mixed vigorously. After fixation the content was kept in room temperature for 1-2 hours. The content was again centrifuged at 1500 rpm and supernatant was removed and this step was continued until pellet becomes white. For the preparation of slides the new slides were first refrigerated and then cell button mix was dropped over the slides and dried immediately on a hot plate, and then was kept in an incubator for proper drying. The slides were then placed in a coplin jar containing Giemsa staining for 4 minutes and destained in a coplin jar containing distilled water for 1 minute. The slides were dried and then viewed under microscope for stained chromosome. . The slides were then viewed under 100X power under oil immersion objective of the microscope to analyze the chromosome aberration s. Onion Root Tip SCE Assay The onion root tips[1], 2-3 cm long, were soaked in 100  µM 5-bromodeoxy uridine (BrdUrd) for almost 20 h followed by 1 hour treatment with the crude extract After a brief wash, the roots were allowed to grow for another round in growing media. The treatments were terminated by washing the roots with distilled water and then 0.05% Colchicine was added then incubated for 2.5 h. Roots were washed, excised and fixed in Carnoy’s fixative, for 1-3hrs and preserved at 4 °C. The roots were processed using cytology methods for SCE analysis.. The roots were then hydrolysed in 5 N HCI at 25 °C for 92 min and stained with haematoxylin for at least 2hrs. The stained root[16] were washed in distilled water, squashed in a drop of 45% acetic acid and tapped for metaphase chromosome separation under coverslips. Tap water controls were included in the assay. The slides were observed at 100X magnification in oil immersion using light microscope RESULTS AND DISCUSSION Dragendorff’s test Fig 1: Showing result of sample after Dragendorff’s test The alkaloids present in the puffer fish was precipitated as a complex formation by dragendorff’s reagent. Dragendorff’s test results showed very high precipitation in skin and intestine, high precipitation in liver and very low precipitation or almost no precipitation was observed in kidney, gonads and eye. Gas-Chromatography-Mass Spectrometry Characteristic peak was observed at retention time 8.33 and 8.66 in liver, intestine and skin after performing alkaline treatment and there was no characteristic peak observed in kidney, eyes and gonads. After boiling of samples which contain TTX in alkaline solution (NaOH) the compound TTX present gets reduced to C9 base TMS (trimethysilyl). It is noteworthy that each peak of selected ion monitored at m/z = 376, 392 and 407 appears at the same retention time in the Selected ion-monitored mass chromatogram of the TMS derivatives of alkali-hydrolyzed. From samples of liver, kidney and intestine, mass fragments of ion peaks was observed at ion M/z 376, 392 and 407, which are characteristic of the quinazoline skeleton (C9 base), which was almost similar as those from the TMS-C9 Base derived authentic TTX Fig 2: Showing GC-MS spectrum of the TMS derivatives of alkali-hydrolysed toxin from Diodon hystrix UV-spectroscopy In UV analysis method characteristic peaks were observed in all samples. Shoulder peak was observed in liver, intestine and skin, Declining and Inclining Peaks were observed in kidney, eyes and gonads. The UV spectrum is analyzed for the characteristic of absorptions, associated with C9-base .The shoulder peaks were observed at 276 nm indicates the formation of C-9 base which were specific to TTX or related substances. Fig 3: Showing chart of UV-spectroscopy of the crude extract from various organs of Diodon hystrix, peak at 276nm indicating the presence of TTX. Genotoxicity Human Leukocyte Assay Metaphase plates were obtained while observing under 100X magnification in oil immersion using light microscope. It has been observed in all the samples that there were no chromosomal aberration that is structural or numerical chromosomal modification were not observed. From this result, it can be reported that the crude extract from Diodon hystrix has no clastogenic (breakage of chromosome) or aneugenic (change in chromosomal number) effects. Fig4(left): Showing metaphase plate from control leukocytes. Fig5(right):  Showing metaphase plate from crude extract leukocytes. Onion Root Tip SCE Assay The Sister Chromatid Exchange (SCE) assay has been reported to be one of the most sensitive short-term genotoxicity assays because of its capability to identify genotoxins at very low doses (Tucker et al.1993). It has been observed that the crude extract from Skin and intestine enhanced SCE significantly over the control while the Liver, Eye, Gonads and Kidney have very low effects. Therefore it can be put forth that the crude extract from skin and intestine interfere to a great deal with the SCE and further studies need to be carried out. Fig6(left) : Showing result of SCE in control onion root tip. Fig7(right): Showing result of SCE in crude extract root tip. Conclusion: From the study, it can be reported that Diodon hystrix from the eastern coastal region of India, observed to have accumulated TTX in its organs. Thus it can be toxic when ingested and even lethal to the predators. Nevertheless further studies should be carried out on this fish to confirm the presence of a homologue of TTX and obtain a purified sample of the TTX. References: Samanta S.Khora, Kamal K.Panda and Brahma B.Panda (1997): Genotoxicity of tetrodotoxin from puffer fish tested in root meristem cells of Allium cepa L. 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