Rajeshwar N. Sharan

Association of Betel Nut with Carcinogenesis: Revisit with a Clinical Perspective

Abstract Betel nut (BN), betel quid (BQ) and products derived from them are widely used as a socially endorsed masticatory product. The addictive nature of BN/BQ has resulted in its widespread usage making it the fourth most abused substance by humans. Progressively, several additives, including chewing tobacco, got added to simple BN preparations. This addictive practice has been shown to have strong etiological correlation with human susceptibility to cancer, particularly oral and oropharyngeal cancers. The PUBMED database was searched to retrieve all relevant published studies in English on BN and BQ, and its association with oral and oropharyngeal cancers. Only complete studies directly dealing with BN/BQ induced carcinogenesis using statistically valid and acceptable sample size were analyzed. Additional relevant information available from other sources was also considered. This systematic review attempts to put in perspective the consequences of this widespread habit of BN/BQ mastication, practiced by approximately 10% of the world population, on oral cancer with a clinical perspective. BN/BQ mastication seems to be significantly associated with susceptibility to oral and oropharyngeal cancers. Addition of tobacco to BN has been found to only marginally increase the cancer risk. Despite the widespread usage of BN/BQ and its strong association with human susceptibility to cancer, no serious strategy seems to exist to control this habit. The review, therefore, also looks at various preventive efforts being made by governments and highlights the multifaceted intervention strategies required to mitigate and/or control the habit of BN/BQ mastication.

INFLUENCE OF HISTONE ACETYLATION ON THE MODIFICATION OF CYTOPLASMIC AND NUCLEAR PROTEINS BY ADP-RIBOSYLATION IN RESPONSE TO FREE RADICALS

Inhibition of histone deacetylase by addition of 5 mM n-sodium butyrate to the growth medium increases the utilization of [32P]NAD+ and ADP-ribosylation (ADPR) of total cellular proteins of V79, HeLa, mouse B16, mouse Fib/T and human T1 kidney cells by a factor of 1.2-2.3. When the ADP-ribosylase is challenged by exposing cells to damage by .OH radicals (25 microM CuSO4 2.8 mM H2O2) ADPR increases by factors of 5.7-6.0 and 3.2-4.0 in normal and butyrated cells, respectively. Operation of the free radical generator is supported by the response to EDTA and radical scavengers. Densitometric analysis of autoradiographs from SDS-gels show that butyrate exposure increases basal ADPR-modification of histones from T1 cells by factors of 1.1-1.9. Addition of .OH radicals increases the ADPR modifications of histones 4.4-8.7-fold in normal cells and 3.2-6.7-fold in butyrate exposed cells. Butyrate exposure elevates base level ADPR-modification and reduces subsequent ADPR-modification initiated by DNA damage. The results are consistent with the view that ADPR-modification and histone acetylation have overlapping functions and probably induce similar structural changes in chromatin.

Proteomic and genomic modulations induced by γ-irradiation of human blood lymphocytes

Purpose: Quantitative evaluation of early response proteins (ERPRO) and early response genes (ERG) following γ-irradiation of human lymphocytes; identification of specific proteins and genes as candidate biomarkers for the development of a novel biodosimeter. Materials and methods: Human peripheral blood lymphocytes were exposed to clinically relevant doses (1, 2 and 4 Gy) of γ-radiation ex-vivo. Analyses of protein and gene expression modulation were conducted 2 h post-irradiation. Global modulations were monitored using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and DNA microarray analyses of the samples originating from one human donor. On the proteome level, both phosphorylated and non-phosphorylated proteins were considered. Proteins and genes of specific interest were further targeted using Western blot (WB) and real-time quantitative polymerase chain reaction (RT-qPCR) techniques, employing samples from several human donors (n = 3). Results: A set of ERPRO and ERG showing significant alterations 2 h post-γ-irradiation have been identified in human lymphocytes. The most radiation responsive genes and proteins indicated alterations of cellular structure (β-actin, talin-1 [TLN1], talin-2, zyxin-2), immune and defence reactions (major histocompatibility complex binding protein-2 [MBP2], interleukin-17E and interferon-γ), cell cycle control (cyclin-dependent kinase inhibitor-1A [CDKN1A], mouse double minute-2, annexin-A6 [ANXA6], growth arrest and DNA-damage-inducible protein-α [GADD45A], proliferating cell nuclear antigen [PCNA], dual specificity phosphatase-2 and 8 [DUSP8]) as well as detoxification processes (peroxin-1) and apoptosis (B-cell lymphoma-2 binding component-3 [BBC3]). Summary: The estimations of protein concentration modulation of TLN1 and CDKN1A, phosphorylation status of ANXA6 (dose range 0–2 Gy) and MBP2 as well as the alterations in the level of gene expressions of BBC3, DUSP8, GADD45A and PCNA appears to be of potential value for future biodosimetric applications.

211At-αDose Dependence of Poly-ADP-Ribosylation of Human Glioblastoma Cells in VitroSuitability in Cancer Therapy?

Aim: It was intended to test the biological response (poly-ADP-ribosylation of cellular proteins) of a-particles from extracellular 211At for enhanced damage to human glioblastoma cells in vitro and to discuss its suitability for potential application in therapy of high-grade gliomas. Materials and Methods: Confluent cultures of human glioblastoma cells were exposed to different doses of a-radiations from homogeneously distributed extracellular 211At. Cellular poly-ADP-ribosylation of all proteins including histones was monitored since it is an indirect but sensitive indicator of chromatin damage and putative repair in both normal and malignant mammalian cells. Results: A significant diminution (average 85.6%) in poly-ADP-ribosylation of total cellular proteins relative to that for non-irradiated glioblastoma cells was observed following 0.025 to 1.0 Gy a-radiations. In the dose range of 0.0025 to 0.01 Gy there was an increase with a maximum value of approximately 119.0% at 0.0025 Gy. Below 0.0025 Gy no change in poly-ADP-ribosylation was observed. Conclusions: Level of cellular poly-ADP-ribosylation of proteins at 0.025 to 1.0 Gy of a-radiation dose from 211At appears to cause enhanced damage by creating molecular conditions which are not conducive to repair of DNA damages in human glioblastoma cells in vitro. Therefore, it is assumed that clinical application of 211At at least in this dose range might enhance clinical efficacy in radiotherapy of cancer.Ziel: Es war die Absicht, die biologische Reaktion (Poly-ADP-Ribosylierung zellulärer Proteine) menschlicher Glioblastomzellen in vitro auf verstärkte Schadensbildung durch a-Teilchen von extrazellulärem 211At zu testen und deren Berücksichtigung für eine potentielle Anwendung in der Therapie von malignen Glioblastomen zu diskutieren. Material und Methode: Konfluente Kulturen menschlicher Glioblastomzellen wurden unterschiedlichen a-Dosen von homogen verteiltem extrazellulärem 211At ausgesetzt. Die zelluläre Poly-ADP-Ribosylierung aller Proteine, einschließlich die der Histone, wurde bestimmt, da sie ein indirekter, aber empfindlicher Indikator für Chromatinschäden und mutmaßlich für die Reparatur in normalen und entarteten Zellen ist. Ergebnisse: Eine signifikante Verringerung (durchschnittlich 85,6%) der Poly-ADP-Ribosylierung aller zellulären Proteine, relativ zu der der nichtbestrahlten Glioblastomzellen, wurde nach 0,025 bis 1,0 Gy a-Bestrahlung beobachtet. Im Dosisbereich von 0,0025 bis 0,01 Gy gab es einen Anstieg mit einem maximalen Wert von angenähert 119% bei 0,0025 Gy. Unterhalb von 0,0025 Gy wurde keine Änderung der Poly-ADP-Ribosylierung beobachtet. Schlußfolgerung: Das Niveau poly-ADP-ribosylierter Proteine im 211At-a-Dosisbereich von 0,025 bis 1,0 Gy scheint eine erhöhte Schädigung dadurch zu bewirken, daß molekulare Bedingungen geschaffen werden, die der Reparatur von DNA-Schäden in menschlichen Glioblastomzellen in vitro nicht förderlich sind. Daher wird angenommen, daß die klinische Anwendung von 211AT – zumindest in diesem Dosisbereich – die Wirksamkeit der Radiotherapie von Krebs steigern könnte.

Altered BRCA1 and BRCA2 responses and mutation of BRCA1 gene in mice exposed chronically and transgenerationally to aqueous extract of betel nut (AEBN).

The Brca1 and Brca2 tumor suppressor genes are involved in the maintenance of genomic integrity as they facilitate error free DNA repair. This study was designed to understand the role of Brca1 and Brca2 in betel nut (BN) induced chronic and transgenerational carcinogenesis in mice. Young male and female Swiss Albino mice were chronically as well as transgenerationally exposed to aqueous extract of betel nut (AEBN) in drinking water (2 mg ml(-1)) for up to 24 weeks. In chronically exposed mice, the levels of Brca1 and Brca2 proteins were elevated to approximately 1.4-fold over the age matched controls after 2 weeks of exposure to AEBN, followed by a decline below the controls. In transgenerationally exposed mice, both Brca1 and Brca2 proteins remained below the controls from the onset of AEBN exposure and rapidly declined further, indicating a loss of tumor suppressor protection. Nucleotide sequencing of exon 11 of Brca1 and exon 27 of Brca2 did not reveal mutation in liver nodules of chronically exposed mice, while a G → C mutation Brca1 was observed in liver nodules as well as in solid tumors developing in transgenerationally exposed mice. Thus, the genomic instability arising due to the lowering in the levels of Brca1 and Brca2 proteins and mutation in exon 11 of Brca1 gene contributed to the increased risk of cancer in mice exposed transgenerationally to AEBN.

Detection and quantification of poly-ADP-ribosylated cellular proteins of spleen and liver tissues of mice in vivo by slot and Western blot immunoprobing using polyclonal antibody against mouse ADP-ribose polymer

Poly-ADP-ribosylation (PAR) of cellular proteins has been shown to have decisive roles in diverse cellular functions including carcinogenesis. There are indications that metabolic level of poly-ADP-ribosylated cellular proteins might indicate carcinogenesis and, therefore, could be potentially used in cancer screening program. Keeping in mind the limitations of currently available assays of cellular PAR, a new assay is being reported that measures the metabolic level of poly-ADP-ribosylated cellular proteins. The ELISA based slot and Western blot immunoassay used polyclonal antibody against natural, heterogeneous ADP-ribose polymers. It could be successfully employed to qualitatively and quantitatively assay metabolic levels of poly-ADP-ribosylated proteins of spleen and liver tissues of normal mice or mice exposed to dimethylnitrosamine for up to 8 weeks; potentially PAR of cellular proteins could be assayed in any tissue or biopsy. Implications of the results in cancer screening program have been discussed. (Mol Cell Biochem 278: 213–221, 2005)

Altered p53 response and enhanced transgenerational transmission of carcinogenic risk upon exposure of mice to betel nut.

Alteration of p53 protein level, and possible mutation of the p53 gene during carcinogenesis in mice exposed chronically (P) and transgenerationally to 2mg/ml aqueous extract of betel nut (AEBN) in drinking water, were studied. Exons 5 and 7 of the p53 gene were not mutated under both chronic and transgenerational exposure, but, p53 protein response was altered. In P mice, p53 protein was initially upregulated in comparison to age-matched controls, reaching 2.5 folds in the liver after 6 weeks of exposure. Subsequently, p53 protein declined to control level after 16 weeks, with concomitant preneoplastic nodulation of the liver. After 24 weeks, p53 protein was below control level, and preneoplastic nodules were well-developed. The level of p53 protein in transgenerationally exposed mice remained invariant in comparison to age-matched controls. Liver nodulation was significantly advanced, developing in F1 mice after 8 weeks, F2 mice after 6 weeks and F3 mice after 4 weeks of exposure. Anomalies not observed in P mice, developed in transgenerationally exposed mice, albeit, non-significantly. Thus, AEBN exposure enhanced transgeneration transmission of carcinogenic risk.

In vivo exposure of Swiss albino mice to chronic low dose of dimethylnitrosamine (DMN) lowers poly-ADP-ribosylation (PAR) of bone marrow cell and blood lymphocyte proteins

Efforts to identify an easy and convenient biomarker of carcinogenesis with potentials of application in mass screening program continue. In a series of investigations on mice exposed to different carcinogens, poly-ADP-ribosylation (PAR) of cellular proteins of different tissues has been shown to be a potential biomarker of carcinogenesis. Because blood based biomarker of carcinogenesis offers significant advantage in its use in a cancer screening program, this investigation was undertaken to find correlations between initiation of carcinogenesis and PAR of bone marrow cell (BMC) and blood lymphocyte (BL) proteins in mice chronically exposed to low dose of dimethylnitrosamine (DMN) for up to four weeks in vivo. The exposure was either alone or in combination with 3-aminobenzamide (3-AB), an inhibitor of PAR. Total PAR of cellular proteins and of histone H1 protein were monitored by slot and Western blot immunoprobe assays, respectively. The PAR of total cellular proteins as well as of histone H1 was down-regulated in duration of exposure dependent manners. The results suggest that BMC and BL mirrored status of PAR in other tissues. This finding opens up the possibility of using PAR as a biomarker of carcinogenesis in a blood based test utilizing immunoprobe assay of cellular PAR.

Qualitative change in mice liver HMG proteins after low dose chronic administration of aqueous extract of betel nut and diethylnitrosamine

Abstract High mobility group (HMG) proteins have roles in the organization and function of chromatin. Their involvement in carcinogenesis is, however, not clear. Swiss albino mice were exposed to aqueous extract of betel nut (AEBN) and a hepatocarcinogen, diethylnitrosamine (DEN), in a chronic low dose protocol for up to 4 weeks. Studies of their effects on the liver HMG proteins during the process showed that major HMG proteins 1, 2 14 and 17 were qualitatively affected. HMG proteins of the treated animals were found to elute earlier from CM-sephadex column than that of the control. Conformational changes in the HMG proteins under the influence of these carcinogens were observed. The results obtained in this investigation indicate that both carcinogens induced similar changes in HMG proteins of liver of mice.

Ultrastructural alterations in liver of mice exposed chronically and transgenerationally to aqueous extract of betel nut: Implications in betel nut-induced carcinogenesis

The aqueous extract of betel nut (AEBN) induces the formation of preneoplastic nodules in the liver of Swiss Albino mice and leads to increased predisposition to cancer when administered transgenerationally. The aim of this investigation was to elucidate the alterations in ultrastructure of subcellular organelles in the liver nodules using transmission electron microscopy and to determine whether these alterations have implications in AEBN‐induced carcinogenesis. Male and female Swiss Albino mice were exposed to AEBN chronically and transgenerationally at a dose of 2 mg/mL in drinking water for 24 weeks. Extensive polymorphism was noted in nuclear shape and heterochromatin organization. Heterochromatin aggregation and marginalization were observed in the nuclei of chronically exposed mice, whereas transgenerationally exposed mice exhibited dispersion or loss of heterochromatin. The nuclear envelope was disrupted, and the nucleoli were enlarged in chronically exposed mice, whereas in transgenerationally exposed mice the nucleoli were reduced in size or totally absent. The cisternae of the rough endoplasmic reticulum were dilated and disrupted, and a large number of autophagic vesicles were observed in both chronically and transgenerationally exposed mice. Atypical mitochondria that underwent extensive cristolysis and progressively declined in size and number from the chronically exposed mice to the different generations of transgenerationally exposed mice were also observed. Thus, exposure to AEBN resulted in severe loss of ultrastructural integrity of cells in the liver nodules, and the progressive loss of mitochondrial function appeared to play a significant role in increasing the predisposition to cancer of mice exposed transgenerationally to AEBN. Microsc. Res. Tech. 2010.