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The Good, the Bad and the Ugly Faces of Areca Nut Mastication

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The Good, the Bad and the Ugly Faces of Areca Nut Mastication The good, the bad and the ugly faces of Areca nut mastication PROF. R. N. SHARAN DEPARTMENT OF BIOCHEMISTRY NORTH-EASTERN HILL UNIVERSITY SHILLONG 793 022 INDIA [email protected] Areca catechu L. (Arecaceae family) 2 A native tropical crop of South Asia ¡ Malaysia, Vietnam, Philippines, etc. ¡ Mostly coastal area Relocated to many countries ¡ Indian subcontinent, West Indies, South Africa, China, etc. ÷ India is largest producer of betel nut in the world! Has been an integral part of the social and religious customs in India ¡ Strong association with betel (paan) leaf (Piper betle) and lime! ÷ Popularly called the “betel nut (BN)”, especially in India Why and how? Workshop on AN & SLT, NICPR 27th November 2017 The good… 3 Areca plant – a cash crop! o Economic value Multitude of usage ¡ Medicine, timber, fuel wood, vegetable, clothing, lubricant, tannin, etc. Medicinal properties ¡ Referred to as “sanjeevani” in some Indian scriptures! ¡ Arecoline, an alkaloid of betel (Areca) nut ÷ CNC stimulant, alertness, a sense of well-being, euphoria, feel-good ÷ Saliva stimulant – appetizer & aids digestion ÷ Mouth refresher ÷ Wormicidal Workshop on AN & SLT, NICPR 27th November 2017 The good… 4 Described widely in ancient Indian scriptures ¡ Vedic Sanskrit shlokas describes ‘good’ of betel (Areca) nuts ÷ Possessing 13 qualities found in heavens! Widely used in our religious rituals… Various names in different parts of the world ¡ Betel nut/Areca nut/Supari/Tambul/Kwai/Pinlang/Pinang… Workshop on AN & SLT, NICPR 27th November 2017 The bad… 5 Weakly addictive Estimated masticators ∼700 million individuals worldwide Rampant and undisciplined usage - a norm today! One of the most commonly used (misused?) masticators in India! Fourth most commonly used (misused?) substance/drug in the world!! ¡ Tobacco > Alcohol > Caffeine > Betel Nut IARC 1985, 2004; RNS: Cancer L., 1996; RNS& YC: In 2010; RNS et al.: PLoS One 2012 Workshop on AN & SLT, NICPR 27th November 2017 The bad… 6 Always masticated with add-ons: QUID ¡ Normal add-ons ÷ Betel leaf, lime, catechu, etc. ¡ Additional add-ons ÷ Smokeless tobacco (SLT)/Chewing tobacco (CT) ¢ In various forms ÷ Colors ÷ Flavors/Perfumes ÷ Sweeteners ÷ Other ingredients Areca nut remains the dominant partner of a quid! Workshop on AN & SLT, NICPR 27th November 2017 The bad… 7 Health hazard of spitting ¡ Spread of infections Social aspects ¡ Loss of man-hour Aesthetic aspect ¡ Widespread nuisance due to spitting ÷ Filth in corners! Exploitation of gender inequality! ÷ Kwai/Tambul sellers in NER ÷ Betel beauties of Taiwan, etc. ¢ School dropping out of young girls! Workshop on AN & SLT, NICPR 27th November 2017 The ugly… 8 An acquired and totally avoidable life-style factor showing strong association with cancer! Increasing trend of AN mastication in combination with SLT/CT!! ¡ Is AN a ‘driver’ to SLT/CT addiction? The habit of Areca nut (AN) chewing ¡ Strong association with cancers of the mouth, oropharyngeal cavity, and upper digestive tract in humans ¡ Cancer capital of India – Mizoram, a city in the north-eastern part of India ¡ GATS 2 (2016-17) report: Record 2-3 fold higher tobacco prevalence among youth (15-17 years) in NER than Indian average! Workshop on AN & SLT, NICPR 27th November 2017 The ugly… 9 AN – a ‘Group 1’ carcinogen by IARC (WHO, 2012) Addition of SLT/CT is like adding fuel to fire! ¡ Two to tango? Asia/Pacific region: AN qualifies to be considered a ‘legal drug’ with a social sanctions! IARC: Similar ‘adjusted’ risk of cancer to humans ¡ From AN alone or AN in combination with SLT/CT ÷ Over-kill effect? Workshop on AN & SLT, NICPR 27th November 2017 The ugly continues… 10 RNS & YC: In ‘Environ. Fact., Gene. & Develop. Human Cancer’, 2010 Workshop on AN & SLT, NICPR 27th November 2017 Dry & wet variety of Areca nut … 11 Dry variety Wet variety Ø Very hard nut of ~ 2 cm diameter Ø Relatively soft nut of ~ 2 cm diameter Ø Usually cut into 4 - 8 or more pieces Ø Usually cut into 4 pieces Ø Ripe (yellowish) or unripe (greenish) nuts Ø Unripe (greenish) nuts from plant Ø Sun-dried directly for several weeks Ø Shell or fibrous coat removed Ø Sun-dried after ‘curing’ or ‘processing’ Ø Shell or fibrous coat removed after Ø Shell or fibrous coat removed ‘ripening’ Workshop on AN & SLT, NICPR 27th November 2017 Research on Areca nut carcinogenesis 12 Swiss albino mice model Experimental mode of application of AN ¡ Nut or powder ¡ Betel nut extract (BNE) ¡ Primary alkaloid, arecoline ¡ Their nitroso derivatives (BN specific nitrosamines) The genotoxic and cytotoxic effects of AN documented Arecoline ¡ Rapid depletion of intracellular thiols ¡ DNA strand breaks ¡ Unscheduled DNA synthesis (UDS) ¡ Mutations ¡ Enhanced rate of cell proliferation ¡ Abnormality in the shape of sperm heads ¡ General embryonic developmental retardation ¡ Teratogenic Workshop on AN & SLT, NICPR 27th November 2017 Constituents of AN Areca nut Alkaloids (Reduced pyridines) ARECOLINE ARECAIDINE 1,2,4,5,-tetrahydro-1- 1,2,5,6,-tetrahydro-1- GUVACINE GUVACOLINE methyl-pyridinecarboxylic methyl-3-pyridinecarboxylic Methyl ester of Methyl ester of acid acid 155.19 kDa 141.17 Da Arecaidine Guvacine Polyphenols Hexahydroxyfla Catechin Flavanoids Leucocyanidins vans Tannins? Gallotannic Gallic acid D-catechol Phiobotannin acid Workshop on AN & SLT, NICPR 27th November 2017 Constituents of AN Constituent Dry variety (%) Wet variety (%) Total 0.25 0.28 – 0.44 Arecoline 0.15 0.18 - 0.24 Alkaloids Arecaidine 0.10 0.10 – 0.20 Others Trace 0.14 Polyphenols 15 23 Tannins 18 22 Carbohydrates 25 30 Proteins 7.5 12 Fats 1.2 2.5 Fibre 15 18 Water Low High Ash content Low High IARC monographs; RNS: Cancer J., 1996 Workshop on AN & SLT, NICPR 27th November 2017 Metabolic activation via P450… 15 AN cellular toxicity: (a) Metabolic activation via ‘nitrosation’ (b) Reversible SUGGESTION: Positive exploitation possible!? KKW & RNS: Int. J. Cancer, 1991 Workshop on AN & SLT, NICPR 27th November 2017 BSNA… 16 BSNA on DNA Dose and exposure period dependent Dose dependent increase in molecular size – gel retardation * * * * * * * * * Estimate: Chronic exposure to AEBN adds 1 BNSA per ~ 7 NT (~ one helical turn of DNA helix) CB & RNS: Mol. Med. Reports, 2008 Workshop on AN & SLT, NICPR 27th November 2017 DNA strand breaks and UDS … 17 Strand breaks: Arecoline > AEBN UDS induction: Arecoline ∼ AEBN Survival: Arecoline < AEBN SUGGESTION: Qualitative difference in cellular response; positive exploitation possible!? KKW & RNS: J. Cancer Res Clin Oncol., 1988; RNS & KKW: Mutat. Res., 1992 Workshop on AN & SLT, NICPR 27th November 2017 Interactions with proteins … 18 AN metabolic toxicity via: (a) Interference with metabolically active proteins (b) Conformational change induction SUGGESTION: Protein associated adduct formation RNS: Cancer J., 1996, 1998; TP & RNS: Hepatol. Res., 1998 Workshop on AN & SLT, NICPR 27th November 2017 Poly-ADP-ribosylation (PAR) … 19 Ø A post-translational modification of primarily chromosomal proteins Ø ADP-ribose moieties from endogenous NAD+ are covalently bound to the target proteins adding ADP-ribose polymer adduct (ARPA) on target protein Ø Carcinogenesis, gene expression, replication, DNA damage and repair, etc. are some cellular events influenced Ø ELISA-based slot- and Western-blot immunoassay Ø ARPA of proteins in any tissue Ø Specific, convenient, non-radioactive and sensitive Workshop on AN & SLT, NICPR 27th November 2017 AEBN & ARPA in mice tissues… 20 Coomassie stained Immunoprobed C 1 2 3 4 C 1 2 3 4 C 1 2 3 4 Slot blot: ARPA of total proteins – AEBN induced Western blot: ARPA of individual initiation in mice; Spleen protein – AEBN induced initiation in cells mice; Spleen cells RNS: In, Adv. Radiat. Cancer Biol., 1998; RNS et al.: Mol. Cell. Biochem., 2005 Workshop on AN & SLT, NICPR 27th November 2017 ARPA in mice blood lymphocytes … 21 120 AEBN 100 80 60 40 PAR (mean +/- (mean PAR SD) 20 0 0 1 2 3 4 Period of exposure (Week) RNS: In, Adv. In Radiat. Cancer Biol., 1998; LK & RNS: Proc. Natl. Acad. Sci. India, 2003; JD & RNS: Cancer Detect. Prev., 2005; JD & RNS: Cancer Letts. 2005 Workshop on AN & SLT, NICPR 27th November 2017 ARPA of PBL in human cancers … 22 Workshop on AN & SLT, NICPR 27th November 2017 ARPA of PBL in human cancers … 23 Workshop on AN & SLT, NICPR 27th November 2017 Extended trans-generational study 24 Swiss albino mice Chronic exposure protocol ¡ Aqueous betel nut extract (AEBN) ¡ 2 mg ml-1 in drinking water Parameters studied ¡ Ultra-structural studies ÷ Histopathology and TEM ÷ Damage to cell and cell organelles ÷ Mitochondrial indices ¡ Tumor Suppressor Genes ÷ TP53 ÷ BRCA1 ÷ BRCA2 ¡ Cellular levels ¡ Mutational status Workshop on AN & SLT, NICPR 27th November 2017 TEM: Liver nucleus… 25 C P P F1 F2 F3 Workshop on AN & SLT, NICPR 27th November 2017 TEM: Liver ER… 26 C P P F1 F2 F3 Workshop on AN & SLT, NICPR 27th November 2017 TEM: Liver mitochondrial structure… 27 C P F1 F2 F3 Workshop on AN & SLT, NICPR 27th November 2017 TEM: Liver mitochondrial number… 28 C P F1 F2 F3 Workshop on AN & SLT, NICPR 27th November 2017 Progressive down-regulation of ‘Mitochondrial Index’ (MI) 29 2500 Control P SD ± 2000 F1 F2 1500 F3 1000 500 area of mitochondria) mitochondria) of area Mitochondrial X (Number index Mitochondrial 0 Control P F1 F2 F3 Generation Workshop on AN & SLT, NICPR 27th November 2017 Conclusions 30 Loss of organelle functions due to destruction of ultrastructure from P through F3 generations Progressively decreasing MI from P through F3 generations
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