Project description:Viral hepatitis and aflatoxin B1 (AFB1) exposure are common risk factors for hepatocellular carcinoma (HCC). The incidence of HCC in individuals co-exposed to hepatitis C (HCV) or B virus and AFB1 is greater than could be explained by the additive effect, yet the mechanisms are poorly understood due to lack of an animal model. This study investigated the outcomes and mechanisms of combined exposure to HCV and AFB1. We hypothesized that HCV transgenic (HCV-Tg; expressing core, E1, E2, and p7, nucleotides 342-2771) mice will be prone to hepatocarcinogenesis when exposed to AFB1. Neonatal (7 days old) HCV-Tg or C57BL/6J wild-type mice were exposed to AFB1 (6 ug/g bw) or tricaprylin vehicle (15 ul/g bw) and male offspring were followed for up to 12 months. No liver lesions were observed in vehicle-treated wild type or HCV-Tg mice. Tumors (adenomas or carcinomas) and preneoplastic lesions (hyperplasia or foci) were observed in 22.5% (9 of 40) of AFB1-treated wild-type mice. In HCV-Tg, the incidence of tumorous or pre-tumorous lesions was significantly elevated (50%, 18 of 36), with the difference largely due to a 2.5-fold increase in the incidence of adenomas (30.5% vs 12.5%). While oxidative stress and steato-hepatisis were observed in both AFB1-treated groups, molecular changes indicative of the enhanced inflammatory response and altered lipid metabolism were more pronounced in HCV-Tg mice. In summary, HCV proteins core, E1, E2 and p7 are sufficient to reproduce the additive co-carcinogenic effect of HCV and AFB1 which is a known clinical phenomenon. HCV transgenic mice (SL-139 strain, pAlbSVPA-HCV-S, containing the structural genes core, E1, E2, and p7, nucleotides 342-2771 of HCV genotype 1b, strain N, under the control of the murine albumin promoter/enhancer) on C57BL/6J (Jackson Laboratory, Bar Harbor, ME) background were previously reported in Lerat et al (Lerat et al., Gastroenterology 122, 352-365, 2002). Transgenic animals were identified after weaning as detailed in Korenaga et al (Korenaga et al., J Biol. Chem. 280, 37481-37488, 2005). Neonatal (7 days old) mice were administered a single dose of AFB1 (6 ug/g bw) or tricaprylin vehicle (15 ul/g bw) by intra-peritoneal injection. Male mice were maintained on the regular animal chow with free access to food and water for up to 12 months. All animal experiments were approved by the UNC Animal Care and Use Committee. There were 20 liver samples used for microarray analysis (12 month time point). All samples were run in one batch. There are 4 groups: WT/Control (4 samples - all biological replicates, i.e., different animals); WT/AFB1 (6 samples); HCV/Control (4 samples); HCV/AFB1 (6 samples). This was a 2 color design with a common reference mRNA. No dye swaps or replicate arrays were included.

Project description:Viral hepatitis and aflatoxin B1 (AFB1) exposure are common risk factors for hepatocellular carcinoma (HCC). The incidence of HCC in individuals co-exposed to hepatitis C (HCV) or B virus and AFB1 is greater than could be explained by the additive effect, yet the mechanisms are poorly understood due to lack of an animal model. This study investigated the outcomes and mechanisms of combined exposure to HCV and AFB1. We hypothesized that HCV transgenic (HCV-Tg; expressing core, E1, E2, and p7, nucleotides 342-2771) mice will be prone to hepatocarcinogenesis when exposed to AFB1. Neonatal (7 days old) HCV-Tg or C57BL/6J wild-type mice were exposed to AFB1 (6 ug/g bw) or tricaprylin vehicle (15 ul/g bw) and male offspring were followed for up to 12 months. No liver lesions were observed in vehicle-treated wild type or HCV-Tg mice. Tumors (adenomas or carcinomas) and preneoplastic lesions (hyperplasia or foci) were observed in 22.5% (9 of 40) of AFB1-treated wild-type mice. In HCV-Tg, the incidence of tumorous or pre-tumorous lesions was significantly elevated (50%, 18 of 36), with the difference largely due to a 2.5-fold increase in the incidence of adenomas (30.5% vs 12.5%). While oxidative stress and steato-hepatisis were observed in both AFB1-treated groups, molecular changes indicative of the enhanced inflammatory response and altered lipid metabolism were more pronounced in HCV-Tg mice. In summary, HCV proteins core, E1, E2 and p7 are sufficient to reproduce the additive co-carcinogenic effect of HCV and AFB1 which is a known clinical phenomenon. HCV transgenic mice (SL-139 strain, pAlbSVPA-HCV-S, containing the structural genes core, E1, E2, and p7, nucleotides 342-2771 of HCV genotype 1b, strain N, under the control of the murine albumin promoter/enhancer) on C57BL/6J (Jackson Laboratory, Bar Harbor, ME) background were previously reported in Lerat et al (Lerat et al., Gastroenterology 122, 352-365, 2002). Transgenic animals were identified after weaning as detailed in Korenaga et al (Korenaga et al., J Biol. Chem. 280, 37481-37488, 2005). Neonatal (7 days old) mice were administered a single dose of AFB1 (6 ug/g bw) or tricaprylin vehicle (15 ul/g bw) by intra-peritoneal injection. Male mice were maintained on the regular animal chow with free access to food and water for up to 12 months. All animal experiments were approved by the UNC Animal Care and Use Committee.

Project description:Aflatoxin B1 (AFB1) is extremely carcinogenic and can cause liver cancer in humans and animals with continued ingestion. As a natural compound, curcumin (Cur) exhibits excellent anti-inflammatory, and anti-cancer properties with few side effects. In this study, a total of 60 male mice (6-week-olds, 15 per group). After one week of acclimatization feeding, the mice were divided into control group (Con), AFB1 group, curcumin group (Cur), and AF+Cur group. The mice were gavaged with curcumin (Cur, 100 mg/kg) and/or AFB1 (0.75 mg/kg). To identify a new therapeutic target for AFB1-induced pyroptosis, we performed proteomic profiling for curcumin alleviating liver injury caused by AFB1 to further validate the targets through volcano plot analysis, Venn analysis, heatmap analysis, correlation, cluster analysis, GO and KEGG enrichment. AFB1 exposure resulted in the loss of hepatocyte membrane, swelling of the endoplasmic reticulum, and a significant increase in transaminase (ALT and AST) contents, while curcumin greatly improved these changes. We found that differentially expressed proteins are enriched in the endoplasmic reticulum membrane and identified ITPR2 as a target of curcumin that alleviates AFB1-induced liver injury by proteomics. Furthermore, ITPR2 expression was detected by immunofluorescence, and qRT-PCR for mRNA expression of genes downstream of ITPR2 (calpain1, calpain2, caspase-12, caspase-3). ITPR2-activated endoplasmic reticulum stress-related proteins (calpain1, calpaini2, bcl-2, BAX, cl-caspase-12, cl-caspase-3), apoptosis (PARP) and pyroptosis (DFNA5) related proteins were examined by western blotting. The analysis showed that it effectively prevents AFB1-induced pyroptosis by lowering endoplasmic reticulum stress via interfering with ITPR2 and its downstream proteins (calpain1, calpain2, bcl-2, Bax) and inhibiting caspase-12/caspase-3 pathway. Conclusively, this study applied proteomic profiling to elucidate ITPR2 as a new target, which might give a new perspective on the mechanism of curcumin alleviating AFB1-induced pyroptosis.

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Transcriptome analysis of aflatoxin B1 (AFB1) induced hepatocellular carcinoma (HCC) and AFB1 resistant liver sample from rats

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