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)

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— 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…

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— 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…

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— 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…

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— 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…

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RNS & YC: In ‘Environ. Fact., Gene. & Develop. Human Cancer’, 2010 Workshop on AN & SLT, NICPR 27th November 2017 Dry & wet variety of Areca nut …

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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

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— 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…

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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…

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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 …

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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 …

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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) …

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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…

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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)

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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 …

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Workshop on AN & SLT, NICPR 27th November 2017 ARPA of PBL in human cancers …

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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…

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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)

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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

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— Loss of organelle functions due to destruction of ultrastructure from P through F3 generations — Progressively decreasing MI from P through F3 generations — Indicative of progressive loss of apoptosis through generations

YASHMIN CHOUDHURY & R. N. SHARAN (2010) Ultrastructural alterations in liver of mice exposed chronically and transgenerationally to aqueous extract of betel nut: Implications in betel nut-induced carcinogenesis. Microscopy Res. Tech. 73, 530-539.

Workshop on AN & SLT, NICPR 27th November 2017 The TSGs & TSPs…

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— Qualitative and quantitative analyses of cellular levels of tumor suppressor proteins p53, BRCA1 and BRCA2 following AEBN exposure – Slot-blot immunoprobing ¡ Liver, spleen and peripheral blood lymphocytes — Reconfirmed by Western-blot immunoprobing — Under both chronic as well as trans-generational protocols — With age-matched controls for each point — Large study! ¡ Over 350 Swiss albino mice involved in this study

Workshop on AN & SLT, NICPR 27th November 2017 Critical anti-carcinogenesis steps

32 Control of cell proliferation Fidelity of DNA

— Tumor Suppressor Genes (TSG) — Tumor Suppressor Genes (TSG) TP53 BRCA1 & BRCA2 ¡ ‘Gate Keeper’ tumor suppressor ¡ ‘Care Taker’ tumor suppressor proteins (TSP) proteins (TSPs) ¡ P53 ¡ BRCA1 ¡ Cell cycle control ¡ BRCA2 ¡ Efficient DNA repair

Workshop on AN & SLT, NICPR 27th November 2017 p53 in chronically exposed mice – P generation: Qualitative

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Liver Spleen Lymphocytes

Workshop on AN & SLT, NICPR 27th November 2017 Overview p53…

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Liver Spleen Lymphocytes

300 P 300 P P F1 F1 300 250 250 F1 F2 F2 250 F2 200 F3 200 F3 200 F3 150 150 150 100 mean ± SD mean ± mean ± SD mean ± 100

mean ± SDmean ± 100 50 50 50 p53 protein level (percent) p53 protein level (percent) (percent) level protein p53

0 (percent) level protein p53 0 0 0 4 6 8 12 16 24 0 4 6 8 12 16 24 0 (C) 4 6 8 12 16 24 (C) (C) Period of treatment (weeks) Period of treatment (weeks) Period of treatment (weeks)

Workshop on AN & SLT, NICPR 27th November 2017 Overview BRCA1…

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Liver Spleen Lymphocytes

150 P 150 P P F1 F1 150 F2 125 F2 F1 125 F3 F3 125 F2 100 F3 100 100 75 75 75

mean ± SDmean ± 50 50 mean ± SD mean ± 50 mean SD ± 25 25 25 Brca1 protein level (percent) Brca1 protein level (percent) 0 0 Brca1 protein level (percent) 0 0 (C) 2 6 8 12 16 24 0 (C) 2 6 8 12 16 24 0 (C) 2 6 8 12 16 24 Period of treatment (weeks) Period of treatment (weeks) Period of treatment (weeks)

Workshop on AN & SLT, NICPR 27th November 2017 Overview BRCA2…

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Liver Spleen Lymphocytes

150 P 150 P P 150 F1 F1 F1 125 125 F2 F2 125 F2 F3 F3 F3 100 100 100 75 75 75 50 mean SD ±

mean SD ± 50 50 mean ± SD 25 25 25 Brca2 protein level (percent)

Brca2 protein level (percent) 0 0 Brca2 protein level (percent) 0 0 (C) 2 6 8 12 16 24 0 (C) 2 6 8 12 16 24 0 2 6 8 12 16 24 Period of treatment (weeks) (C)Period of treatment (weeks) Period of treatment (weeks)

Workshop on AN & SLT, NICPR 27th November 2017 Conclusions

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— All three TSPs show immediate response following chronic exposure (P generation) — Nil to diminished response of p53, and BRCA1 & BRCA2 with passage of time/generation (F1 through F3) — Progressive down-regulation with progression of generation (P through F3 generation)

Workshop on AN & SLT, NICPR 27th November 2017 Mutational analyses of TSGs

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— TP53, BRCA1 and BRCA2 following AEBN exposure ¡ TP53 ÷ Exons 5 & 7 ¡ BRCA1 ÷ Exon 11 ¢ Five segments ¡ BRCA2 ÷ Exon 27 — Chronic as well as trans-generational protocols

Workshop on AN & SLT, NICPR 27th November 2017 Mutational analyses

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— TP53 ¡ Exons 5 & 7 ÷ No mutation detected! — BARCA1 ¡ Exon 11 ¢ Mutation! — BARCA2 ¡ Exon 27 ÷ Study/analysis underway ¢ Apparently no mutation!

Workshop on AN & SLT, NICPR 27th November 2017 Mutational analysis – Exon 11 – BRCA1 (B fragment)

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A

B

Workshop on AN & SLT, NICPR 27th November 2017 Mutational analysis – Exon 11 - BRCA1 (B fragment)

41 — Point mutation at 41st codon: TGT → TCT — Resulting amino acid change: Cys → Ser — Cys has a –SH group — Ser has an –OH group — The amino acid change may cause three possible rotameric change in the Brca1 protein 3D-structure

Workshop on AN & SLT, NICPR 27th November 2017 Possible consequences of BRCA1 mutation

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— Molecular docking Possibility 1 Possibility 2 — Computer simulation — Swiss PDV Viewer

• NORMAL

• AN EXPOSED

Workshop on AN & SLT, NICPR 27th November 2017 Conclusions

43 — No detectable mutation in TSGs in liver nodules after 24 weeks of exposure to AEBN ¡ Exons 5 and 7 of the p53 gene ¡ Exon 27 of BRCA2 gene — Single G → C transversion type mutation in BRCA1 gene ¡ In segment B of BRCA1 gene – more towards the N-terminal of the TSP ¡ In all trans-generationally exposed mice ¡ F1 through F3 mice — 3D- structural change in Brca1 protein molecule predicted!

Workshop on AN & SLT, NICPR 27th November 2017 Consequences of AN mastication – pre-cancer

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Oral leukoplakia Oral erythroplakia Oral submucous (OL) (OE) fibrosis (OSF)

Workshop on AN & SLT, NICPR 27th November 2017 Consequences of AN mastication…

45 — Oral lichenoid lesion (OLL) — A pre-cancer condition or early cancerous development in the oral cavity — White/red patches with pseudomembranous candidiasis — Lichen, a plant fungi, harboring lesion(s) in the cheek close of the site of mastication — May also manifest in tongue, gums, etc.

Workshop on AN & SLT, NICPR 27th November 2017 Consequences of AN mastication…

46 — Oral lichen planus (OLP) — Late stage of oral lichenoid lesions (OLL) — A type-IV contact hypersensitive lesion

Workshop on AN & SLT, NICPR 27th November 2017 Consequences of AN mastication…

47 — Oral squamous cell carcinoma (OSCC) — A malignant cancer of epithelial lining of the mouth — Keratinization of cells — Fibrosis — Prolonged use of BN alone (without tobacco) — At the primary site of BN mastication

Workshop on AN & SLT, NICPR 27th November 2017 Consequences of AN mastication…

48 — Full blown oral cancer at the floor of the mouth

Workshop on AN & SLT, NICPR 27th November 2017 Cancer predisposition due to AN exposure 49

Period of AN exposure after which Generation pre-neoplastic nodules developed in liver (weeks)

P 16

F1 8*

F2 6**

F3 4***

*Significant difference from P generation at P < 0.05 ** Significant difference from P generation at P < 0.01 *** Significant difference from P generation at P < 0.001

Workshop on AN & SLT, NICPR 27th November 2017 The way forward…

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— Best way forward – QUIT AN mastication! ¡ SLT/CT automatically dropped! — Is it possible? Practical? ¡ Considering the addictive nature of the substance ÷ Is the addictive nature of AN only cause of its widespread usage? ¡ Easy availability of ‘convenient’ alternate forms of the substance ÷ Mouth fresheners/Gutkha/Mawa/Pinlang/Pinang/etc. ÷ Rather inexpensive ¡ Aggressive marketing strategies

Workshop on AN & SLT, NICPR 27th November 2017 The way forward…

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— Next best? ¡ Moderation ¡ Harm reduction! ÷ Alkaloid poor AN? ¡ Segregate AN and SLT/CT mastications!! ¡ Legislating industry standards and its compliance ÷ Quality and quantity control ¡ Strict enforcement of regulations ÷ Price ÷ Availability ÷ Advertisement — Research to fill up gaps in knowledge… Workshop on AN & SLT, NICPR 27th November 2017 Recommendation/Research gap

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¡ Nitrosation: Essential for alkaloid activation & DNA interaction/mutation ÷ Research: Conditions to reduce nitrosation! ¡ Arecoline and AEBN differ in their cellular/molecular actions ÷ Research: Mechanistic understanding ¡ Negative correlation between Arecoline/AEBN and ARPA ÷ Research: Metabolic reversal of the process ¡ Negative trans-generational correlation between Arecoline/AEBN and Mitochondrial Index (MI) with increasing predisposition to cancer ÷ Research: Consequences of altered energy levels & mechanisms ¡ AN and SLT/CT actions: additive or synergistic? ÷ Research: Quantitative and mechanistic understanding

Workshop on AN & SLT, NICPR 27th November 2017 Acknowledgements

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Coworkers Finances

— Dr. Kishore K. Wary — UGC — Dr. Jyoti Rekha Saikia — Dr. B. Jayalata Devi — DST — Dr. Chaitali Bhattacharjee — Dr. M. Meriyani Odyuo — CSIR — Dr. Sanghamitra Chakraborty — Dr. Yashmin Choudhury — NEHU – UPE — Dr. L. Kma — DST - PURSE — Ms. Tongbram Malemnganbi Chanu and — Prof. Ravi Mehrotra — Prof. Kamlesh Asotra

Workshop on AN & SLT, NICPR 27th November 2017 Thank you for your attention…

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