Downloaded from www.microbiologyresearch.org by IP: 54.145.26.59 On: Thu, 14 Apr 2016 15:42:29 Interactions between hepatitis B virus and aflatoxin B 1 : effects on p53 induction in HepaRG cells Myriam Lereau, 1,2 Doriane Gouas, 2 Ste ´ phanie Villar, 2 Ahmad Besaratinia, 3 Agne ` s Hautefeuille, 2 Pascale Berthillon, 1 Ghislaine Martel-Planche, 2 Andre ´ Nogueira da Costa, 2 Sandra Ortiz-Cuaran, 2 Olivier Hantz, 1 Gerd P. Pfeifer, 3 Pierre Hainaut 2,4 and Isabelle Chemin 1 Correspondence Isabelle Chemin isabelle.chemin@inserm.fr Pierre Hainaut pierre.hainaut@cipe.accamargo. org.br Received 25 March 2011 Accepted 21 November 2011 1 INSERM U1052, 151 cours Albert Thomas, 69424 Lyon Cedex 03, France 2 International Agency for Research on Cancer, 150 cours Albert Thomas, 69372 Lyon Cedex 08, France 3 Department of Cancer Biology, Beckman Research Institute, City of Hope Comprehensive Cancer Center, 1500 East Duarte Road, Duarte, CA 91010-3000, USA 4 International Center for Research and Training, Hospital AC Camargo, Rua Pirapitinguı ´ 204, Sa ˜ o Paulo SP 01508 - 020, Brazil Infection by hepatitis B virus (HBV) and dietary exposure to aflatoxin B 1 (AFB 1 ) are the main risk factors for the development of chronic liver disease and hepatocellular carcinoma (HCC). How these factors cooperate is still largely unknown. AFB 1 activation leads to DNA adduction and mutagenesis, with a specific mutation at codon 249 in TP53 (p.R249S). So far, only limited studies have addressed the effects of AFB 1 on HBV replication. We have analysed the effects of both risk factors on p53 induction during HBV infection in HepaRG, a cell line with hepatocyte- like morphology that metabolizes AFB 1 and supports HBV infection. Exposure to AFB 1 up to 5 mM induced a downregulation of HBV replication after 48 h, as measured by a decrease in viral antigens in the culture medium (HBsAg, HBeAg and large envelope protein) and in intracellular levels of HBV transcripts, DNA and HBsAg. Conversely, HBV infection did not significantly modify AFB 1 -DNA adduct formation or repair as assessed by immunodot-blot assay, and the induction of p53 in response to AFB 1 was similar in infected and non-infected HepaRG cells. Overall, our results suggest that AFB 1 exposure decreases HBV replication, whereas DNA damage by AFB 1 and subsequent p53 induction is not affected by the presence of the virus. Thus, in HepaRG cell line, AFB 1 and HBV do not cooperate to increase DNA damage by AFB 1 . Further studies on the effects of both factors in a context of chronicity are needed to better understand synergistic effects. INTRODUCTION Hepatocellular carcinoma (HCC) represents 75–90 % of primary liver cancers (McGlynn & London, 2005). With 694 000 deaths per year in 2008, HCC is the third leading cause of cancer-related deaths (Ferlay et al., 2010). Highest incidences are observed in sub-Saharan Africa and South- east Asia (8–117 per 10 5 in men and 5–75 per 10 5 in women in 2008) (Ferlay et al., 2010). Worldwide, the main risk factors are chronic infections by hepatitis B (HBV) or C (HCV) viruses, accounting for 75–80 % of global HCC cases, often in combination with consumption of foods contaminated by aflatoxin B 1 (AFB 1 ) or with alcohol abuse (Bosch et al., 1999). AFB 1 is classified as a group 1 carcinogen by the International Agency for Research on Cancer (IARC) (WHO, 2002). In areas of highest incidence, HBV is endemic [prevalence of hepatitis B surface antigen (HBsAg) is higher than 8 %] and exposure to AFB 1 is widespread. A synergistic effect of both factors has been observed in cohort studies, with a 5–10-fold higher risk of HCC in the presence of both factors as compared with HBV or AFB 1 alone (Kew, 2003; Lunn et al., 1997; Qian et al., 1994; Ross et al., 1992; Sylla et al., 1999; Wang et al., 1996). HBV is a small hepatotropic virus, belonging to the family Hepadnaviridae, which can cause acute and chronic liver diseases (Ganem & Prince, 2004). HBV genomic DNA, identified as relaxed circular DNA (RC DNA), is partially double-stranded and converted into covalently closed circular DNA (cccDNA) after translocation into the nucleus. cccDNA is the template for viral transcription Supplementary material is available with the online version of this paper. Journal of General Virology (2012), 93, 640–650 DOI 10.1099/vir.0.032482-0 640 032482 G 2012 SGM Printed in Great Britain