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. Mutagenesis vol.12 no.4 pp.265-269 Keyvan Mirbakhsh, Ulf Gà ¶ransson: Tetrodotoxin – evolutionary selection and pain relief Course in Biological Active Natural Products in Drug discovery A8/C, 5p. Distanse course – Fall 2004 Department of Medicinal Chemistry Division of Pharmacognosy Uppsala University. Firoz Ahmed, Aamir Javed, Anup Baranwal, Annu Kumari, Farnaz Mozafari Parvathi Chandrappa (2013):EXTRACTION OF TETRODOTOXIN FROM PUFFER FISH, DIODON LITUROSUS FROM SOUTH ANDAMAN SEA. G.J B.A.H.S., Vol.2 (2) 2013:58-6, ISSN: 2319 – 5584. Teetske F. Van Gorcum, Max Janse, Marianne E.C. Leenders, Irma de Vries, Jan Meulenbelt (2006): Intoxication following minor stabs from the spines of a porcupine fish clinical. Toxicology , 2006, 44(4) p. 391- 393. Vaishali Bane, Mary Lehane, Madhurima Dikshit, Alan O’Riordan and Ambrose Furey (2014): Tetrodotoxin: Chemistry, Toxicity, Source, Distribution and Detection. Toxins 2014, 6, 693-755, ISSN 2072-6651. Bragadeeswaran S, Therasa D, Prabhu K, Kathiresan K (2010): Biomedical and pharmacological potential of tetrodotoxin-producing bacteria isolated from marine pufferfish Arothron hispidus (Muller, 1841). The Journal of Venomous Animals and Toxins including Tropical Diseases ISSN 1678-9199 | 2010 | volume 16 | issue 3 | pages 421-431. J. S. OliveiraI; O. R. Pires JuniorII; R. A. V. MoralesII, III; C. Bloch JuniorIII; C. A. SchwartzII; J. C. FreitasI (2003): Toxicity of puffer fish two species (Lagocephalus laevigatus, linaeus 1766 and Sphoeroides spengleri, Bloch 1785) from the Southeastern Brazilian coast. J. Venom. Anim. Toxins incl. Trop. Dis vol.9 no.1 Botucatu 2003, ISSN 1678-9199. Tamao Noguchi, Kazue Onuki and Osamu Arakawa (2011): Tetrodotoxin Poisoning Due to Pufferfish and Gastropods, and Their Intoxication Mechanism. International Scholarly Research Network ISRN Toxicology Volume 2011, Article ID 276939, 10 pages. Niharika Mandal, Soumya Jal, K. Mohanapriya and S. S. Khora (2013): Assessment of toxicity in puffer fish (Lagocephalus lunaris) from South Indian coast. African Journal of Pharmacy and Pharmacology Vol. 7(30), pp. 2146-2156, ISSN 1996-0816 Md. Moyen Uddin Pk, Rumana Pervin, Dr.Yearul Kabir, Dr. Nurul Absar (2013): PRELIMINARY SCREENING OF SECONDARY METABOLITES AND BRINE SHRIMP LETHALITY BIOASSAY OF WARM-WATER EXTRACT OF PUFFER FISH ORGANS TISSUES, TETRAODON CUTCUTIA, AVAILABLE IN BANGLADESH. Journal of Biomedical and Pharmaceutical Research 2 (5) 2013, 14-18, ISSN: 2279 – 0594 Nagashima, Y., J. Maruyama, T. Noguchi andK. Hashimoto (1987) Analysis of paralyticshellfish poison and tetrodotoxin by ionpairing high performance liquid chromatography.Nippon Suisan Gakkaishi 53:1 819-8 Nakamura, M. and T, Yasumoto (1985)Tetrodotoxin derivatives in puffer fish.Toxicon 23: 271-273 Myoung Ja Lee,Dong-Youn Jeong, Woo-Seong Kim,Hyun-Dae Kim,Cheorl-Ho Kim,Won-Whan Park,Yong-Ha Park,Kyung –Sam Kim,Hyung-Min Kim and Dong –Soo Kim(2000) A tetratoxin –producing Vibrio Strain ,LM-1 from the puffer fish Fugu vermicularisradiates.Appl.Environ.Micribial.Vol.66 no 4 1698-1701 Moorhead, P.S., P.C. Nowell, W.J. Mellman, D.N. Batipps and D.A.Hungerford: Chromosome preparations of leucocytes cultures fromhuman peripheral blood. Exp. Cell. Res., 20, 613-616 (1960). Hungerford, D.A., 1965. Leukocytes cultured fromsmall inocula of whole blood and the preparationof metaphase chromosomes by treatment with Hypotonic KCl. Stain Technol., 40: 333-338. Perry, P. and S. Wolff: New giemsa method for differential staining of sisterchromatids. Nature, 251, 156-158 (1974).

Video recording act 1984 :: essays papers

Video recording act 1984 History As more and more people in the UK started to rent and buy videos for home use, it was thought that some kind of act should be passed that protected the public’s intrest. The video recordings act was brought in to prevent people from making videos of offensive nature (to some people) and then selling that footage to people who are deemed not suitable to see the footage (under aged) or people who are unaware of the nature of the video. In 1984 the act was passed through parliament and the BBFC (British Board Of Film Censorship) was designated to make sure that videos that are offered for sale or hire in the UK have the correct certificate. It was felt that videos needed to have a stricter rating system than cinema films as they could easily be obtained by under aged persons. The act does not really concentrate on the welfare of animals, but does stop films being made which depict scenes of cruelty, sexual (human/animal) intercourse/activities and mutilation. This act does work in relation with other acts that does protect animals, such as the Protection Of Animals Act 1911, Criminal Justice & Public Order and The Performing Animals (regulation) Act 192 5. Act Requirements & Aims: The act affects anyone who wishes to sell or hire videos for home, public use. The act may also effects people who have unlicensed videos for home use (extreme cases) The video recordings act states that: 1) Videos must be classified by designated authority (The president or vice president of the BBFC). That the certificate must reflect the video's suitability for home use. 2) Videos Must Not Contain Graphic Footage of – Â · Torture to humans or animals. Â · Explicit sexual acts between humans or human/animal. Â · Cruelty of animals (as stated in the Protection of Animals Act 1911). Â · Extreme violence to animals or people (particularly children and women). Â · Enjoyment from sexual assaults (human or animal). Â · Scenes that would cause great distress to Animals. These are the main points that effect animals, the rest of the act talks about the classification of the videos and footage in relation to people. 3) That it is an offence to sell such videos to any one under the certificate age. Videos are still allowed to show mild cruelty (depending in what context), for example when they hurl the cow out of the castle in Monty pythons quest for the Holy Grail (15 cert).

Strict Teachers Produce Outstanding Students?

When coming to the discussion whether strict teachers can produce outstanding students, most of us must have our thumbs up and find it absurd and ludicrous if one claimed the famous Chinese saying is wrong. Undoubtedly, strict teachers have high expectation on their students and always demand them to do their best. Under such a strict confinement, students would become more obedient and disciplined. As a result, to a certain extent, students may have a better academic result since they follow what their teachers asked them to do.However, what is the true meaning of â€Å"outstanding†? From my point of view, an outstanding student should not only has good academic result or discipline, but more importantly, balanced development of a student should never be neglected. Therefore, based on the above definition, I cling to the view that strict teachers do not necessarily produce outstanding students. My views will be expressed as follows. First, strict teachers always constrict stu dents' daily activities.Students are requested to spend most of their time studying or doing a host of homework which are assigned by their teachers. Thus, they will have fewer chances to participate in extra-curricular activities or engage in other learning experience. This includes doing exercise or voluntary works, which are vital parts in all-round education. Students' personal development would then be hindered and obviously, they cannot be outstanding on a social dimension. Another standpoint to be put forward is that students' thoughts will be restricted.Nowadays, it is not difficult to notice the significance of critical thinking among students. That's why the latest educational reform stresses on the importance of liberal studies because the previous educational system is too exam-oriented. Yet, strict teachers would ask their students to follow what they have said and refuse all the ideas from students. In this way, students may just follow suit as they are afraid of being punished by their teachers. Hence, they cannot learn how to think critically.This certainly violates the principle of education, let alone producing outstanding students. What is more, the harsh training methods of strict teachers, indisputably, are not suitable for all students. For students who cannot keep abreast of the progess under the harsh training, they may not be able to study efficiently and effectively. In worst case scenario, they might even give up their studies. Being too strict, therefore, cannot guarantee students to be outstanding.It really depends on whether students themselves can accept such kind of teaching methods or not. The teaching methods of strict teachers sound powerful but dreadful at the same time. Strict teachers do not infallibly produce outstanding students. It is essential for them to strike a balance between strict and lenient so diversified development of students can be fostered. I believe that when balanced development can be achieved, an †Å"outstanding† student would be produced.

Pablo Picasso - His Life and His Art Essay - 1222 Words

Pablo Picasso - His Life and His Art Pablo Ruiz y Picasso, painter, sculptor, and printmaker, was born in Malaga Spain on October 25, 1881 and died on April 8, 1973.Today he is considered to be one of the most influential and successful artists in history. Picasso contributed many things to 19th century and modern day art and his name is familiar to all those involved in the many different fields of art. Throughout the seven decades that Picasso produced artwork he used many different types of media. In each piece of art he produced he searched for new possibilities, invented images in them, and reflected events that were occurring in his world through his artwork. Picasso had many artistic influences in his life, including Cà ©zanne,†¦show more content†¦Between 1907 and 1914, Picasso collaborated with Georges Braque and together they produced a style known as Cubism. The period after World War I indicated an apparent return to the art of realism for Picasso as seen in a pencil drawing of 1915, Portrait of Vollard. This small work foretold Picassos renewed curiosity in descrip tive interpretation, which, for some time, ran parallel with cubism. During the 1920s, a growing sense of unease was expressed through Picassos work and a style shift started moving from Cubism to being closer to surrealism (an artistic movement of the early 20th century that stressed fantasy and the subconscious mind). Picassos most well known painting of this time is from 1937, called Guernica. (Grolier). In the last two decades of his life, Picasso became intrigued by earlier art. He based several paintings on works painters of the 1600s such as Rembrandt and Diego Velazquez, and the French artists of the 1800s, such as Delacroix and Manet. Picasso tried to break down their work, recompose it, and became even bolder in his methods and styles of painting (World Book). Picasso influenced artwork of the late 19th and early 20th Century in many ways. He encouraged other artists the explore painting pictures in just one basic colour or one certain colour grouping as most artists of the time painted in a wide spectrum of vibrant melodious colours or pastels. Picassos own exploration of this new idea was seen inShow MoreRelatedArt And The Modern Era And Impact The 20th Century1522 Words   |  7 PagesIntroduction Art is a human activity, the product of this activity or the idea that we are in fact deliberately targeting sense, emotions and intellect. Art and writing are different forms of expression but they both can convey the same intensity for the creator. Art can be used to express thoughts, emotions and feelings whereas writing is a form of expressing ideas and opinions. Pablo Picasso and Ernest Hemingway are those two names, which they don’t need to identification. 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