AFB1-induced Hepatocellular Carcinoma Research Articles

When less is more: The association between the expression of polymorphic CYPs and AFB1-induced HCC.

An individual's genetic fingerprint is emerging as a pivotal predictor of numerous disease- and treatment-related factors. Single nucleotide polymorphisms (SNPs) in drug-metabolizing enzymes play key roles in an individual's exposure to a malignancy-associated risk, such as Aflatoxin B1 (AFB1)-induced hepatocellular carcinoma (HCC). This study aimed at reviewing literature on the polymorphisms that exist in CYP enzymes and their possible link with susceptibility to AFB1-induced HCC. A set of keywords associated with the study subject of interest was used to search the Google Scholar and the PubMed database. The last ten years' worth of research projects were included in the results filter. The research involved HCC patients and any connection between polymorphic forms of CYP enzymes and their susceptibility to AFB1-induced HCC, including older but significant data. Variations in CYP1A2 and CYP3A4 were reported to impact the rate and magnitude of AFB1 bio-activation, thus influencing an individual's vulnerability to develop HCC. In HCC patients, the activity of CYP isoforms varies, where increased activity has been reported with CYP2C9, CYP2D6, and CYP2E1, while CYP1A2, CYP2C8, and CYP2C19 exhibit decreased activity. CYP2D6*10 frequency has been discovered to differ considerably in HCC patients. Rs2740574 (an upstream polymorphism in CYP3A4 as detected in CYP3A4*1B) and rs776746 (which affects CYP3A5 RNA splicing), both of which influence CYP3A expression, thus impacting the variability of AFB1-epoxide adducts in HCC patients. CYP1A2 is the primary enzyme accountable for the formation of harmful AFBO globally. CYP3A4, CYP3A5, CYP3A7, CYP2B7, and CYP3A3 are also implicated in the bio-activation of AFB1 to mutagenic metabolites. It is thought that CYP3A4 is the protein that interacts with AFB1 metabolism the most. Polymorphic variants of CYP enzymes have a functional impact on the susceptibility to AFB1-induced HCC. Outlining such variation and their implications may provide deeper insights into approaching HCC in a more personalized manner for guiding future risk-assessment, diagnosis, and treatment.

Frequencies and spectra of aflatoxin B1-induced mutations in liver genomes of NEIL1-deficient mice as revealed by duplex sequencing.

Increased risk for the development of hepatocellular carcinoma (HCC) is driven by a number of etiological factors including hepatitis viral infection and dietary exposures to foods contaminated with aflatoxin-producing molds. Intracellular metabolic activation of aflatoxin B1 (AFB1) to a reactive epoxide generates highly mutagenic AFB1-Fapy-dG adducts. Previously, we demonstrated that repair of AFB1-Fapy-dG adducts can be initiated by the DNA glycosylase NEIL1 and that male Neil1-/- mice were significantly more susceptible to AFB1-induced HCC relative to wild-type mice. To investigate the mechanisms underlying this enhanced carcinogenesis, WT and Neil1-/- mice were challenged with a single, 4 mg/kg dose of AFB1 and frequencies and spectra of mutations were analyzed in liver DNAs 2.5 months post-injection using duplex sequencing. The analyses of DNAs from AFB1-challenged mice revealed highly elevated mutation frequencies in the nuclear genomes of both males and females, but not the mitochondrial genomes. In both WT and Neil1-/- mice, mutation spectra were highly similar to the AFB1-specific COSMIC signature SBS24. Relative to wild-type, the NEIL1 deficiency increased AFB1-induced mutagenesis with concomitant elevated HCCs in male Neil1-/- mice. Our data establish a critical role of NEIL1 in limiting AFB1-induced mutagenesis and ultimately carcinogenesis.

An in silico toxicogenomic approach in constructing the aflatoxin B1-mediated regulatory network of hub genes in hepatocellular carcinoma

Aflatoxin B1 (AFB1) can cause hepatocellular carcinoma (HCC) through a mutagenic mode of action but can also lead to global changes in gene expression; however, the AFB1 network of molecular pathways involved in HCC is not known. Here, we used toxicogenomic data from human liver cells exposed to AFB1 to infer the network of AFB1-responsive molecular pathways involved in HCC. The following computational tools: STRING, MCODE, cytoHubba, iRegulon, kinase enrichment tool KEA3, and DAVID were used to identify protein-protein interaction network, hub genes, transcription factors (TFs), upstream kinases, and biological processes (BPs). Predicted molecular events were validated with an external dataset, whereas the hub genes in HCC were validated using the UALCAN database. The results revealed an association between AFB1 and the hub genes involved in the cell cycle. We identified TFs that regulate the hub genes and linked them with upstream kinases including cyclin-dependent kinases, mitogen-activated protein kinase 1, and AKT. This approach enabled the construction of the AFB1-mediated regulatory network consisting of upstream kinases, TFs, hub genes, and BPs, thus revealing the signaling hierarchy and information flow that may contribute to AFB1-induced HCC. This could be a useful tool in predicting the molecular mechanisms involved in chemical-induced diseases when available toxicogenomic data exist.

Aflatoxin B1 DNA-Adducts in Hepatocellular Carcinoma from a Low Exposure Area.

Aflatoxin B1 (AFB1) is a class 1 carcinogen with an ascertained role in the development of hepatocellular carcinoma (HCC) in high exposure areas. Instead, this study aimed to assay whether chronic/intermittent, low-dose AFB1 consumption might occur in low-exposure geographical areas, ultimately accumulating in the liver and possibly contributing to liver cancer. AFB1-DNA adducts were assayed by immunostaining in liver tissues from three Italian series of twenty cirrhosis without HCC, 131 HCC, and 45 cholangiocarcinoma, and in an AFB1-induced HCC rat model. CD68, TP53 immunostaining, and TP53 RFLP analysis of R249S transversion were used to characterize cell populations displaying AFB1-DNA adducts. Twenty-five HCCs displayed AFB1-adducts both in neoplastic hepatocytes and in cells infiltrating the tumor and non-tumor tissues. Nuclear immunostaining was observed in a few cases, while most cases showed cytoplasmic immunostaining, especially in CD68-positive tumor-infiltrating cells, suggestive for phagocytosis of dead hepatocytes. Similar patterns were observed in AFB1-induced rat HCC, though with higher intensity. Cholangiocarcinoma and cirrhosis without HCC did not displayAFB1-adducts, except for one case. Despite not providing a causal relationship with HCC, these findings still suggest paying attention to detection and control measures for aflatoxins to ensure food safety in low exposure areas.

Protective and therapeutic effects of sildenafil and tadalafil on aflatoxin B1-induced hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) has been classified as one of the most common forms of liver cancer occurring worldwide, and risk factors include hepatitis B & C virus, alcoholism, and dietary carcinogens like aflatoxin B1 (AFB1), which is produced by fungus Aspergillus flavus and Aspergillus parasiticus. Metabolism of AFB1 resulted into the formation of AFB1-exo-8, 9-epoxide which is largely responsible for HCC development. So far conventional cytotoxic chemotherapy has not provided much benefit in HCC, necessitating the need for newer treatment modalities. Recent reports suggest that phosphodiesterase-5 inhibitors (PDE5i) may have anticancer activity, but till date, the anticancer property of PDE5i (tadalafil & sildenafil) has not been evaluated in HCC. The present study was aimed to define the anticancer property of tadalafil and sildenafil against AFB1-induced HCC rats. Rats were randomly divided into five groups with five rats in each group. Except normal control group, rats of all other groups were fed with 5% alcohol via drinking water for 3weeks. After 3weeks, two successive dose of AFB1 (1mg/kg bw, ip) was administered on subsequent days followed by the administration of PDE5i (tadalafil & sildenafil, 10mg/kg bw) along with drinking water after 6weeks of treatment with AFB1 for 2weeks. An in-depth investigation into its mechanistic aspect revealed that development of HCC induced by aflatoxin B1, decreased the mRNA expression and activity of antioxidant enzyme SOD, GPx, catalase, GR and GST, and GSH content with a concomitant increase in the level of lipid peroxidation. Post-treatment with PDE5 inhibitor (tadalafil & sildenafil) restored the above parameters towards normal, and this result was more effective in case of sildenafil. Thus, results from the above studies suggest that PDE5 inhibitors may act as anticancer agents by preventing the development and progression of HCC by modulating the key parameters of antioxidant pathway.

Aflatoxin B1 induces persistent epigenomic effects in primary human hepatocytes associated with hepatocellular carcinoma.

Chronic exposure to aflatoxin B1 (AFB1) has, in certain regions in the world, been strongly associated with hepatocellular carcinoma (HCC) development. AFB1 is a very potent hepatotoxic and carcinogenic mycotoxin which is frequently reported as a food contaminant. Epigenetic modifications provoked by environmental exposures, such as AFB1, may create a persistent epigenetic footprint. Deregulation of epigenetic mechanisms has actually been reported in HCC patients following AFB1 exposure; however, no attempts have yet been made to investigate early effects on the epigenome level which may be persistent on longer term, thereby possibly initiating carcinogenic events. In this study, we aim to identify methyl DNA-mRNA-interactions representative for a persistent epigenetic footprint associated with the early onset of AFB1-induced HCC. For this, primary human hepatocytes were exposed to 0.3μM of AFB1 for 5 days. Persistent epigenetic effects were measured 3 days after terminating the carcinogenic exposure. Whole genome DNA methylation changes and whole genome transcriptomic analysis were analyzed applying microarray technologies, and cross-omics interactions were evaluated. Upon combining transcriptomics data with results on DNA methylation, a range of persistent hyper- and hypo-methylated genes was identified which also appeared affected on the transcriptome level. For six of the hypo-methylated and up-regulated genes, namely TXNRD1, PCNA, CCNK, DIAPH3, RAB27A and HIST1H2BF, a clear role in carcinogenic events could be identified. This study is the first to report on a carcinogen-induced persistent impact on the epigenetic footprint in relation with the transcriptome which could be indicative for the early onset of AFB1-related development of HCC.

MiR-34a screened by miRNA profiling negatively regulates Wnt/β-catenin signaling pathway in Aflatoxin B1 induced hepatotoxicity.

Aflatoxin-B1 (AFB1), a hepatocarcinogenic mycotoxin, was demonstrated to induce the high rate of hepatocellular carcinoma (HCC). MicroRNAs (miRNAs) participate in the regulation of several biological processes in HCC. However, the function of miRNAs in AFB1-induced HCC has received a little attention. Here, we applied Illumina deep sequencing technology for high-throughout profiling of microRNAs in HepG2 cells lines after treatment with AFB1. Analysis of the differential expression profile of miRNAs in two libraries, we identified 9 known miRNAs and 1 novel miRNA which exhibited abnormal expression. KEGG analysis indicated that predicted target genes of differentially expressed miRNAs are involved in cancer-related pathways. Down-regulated of Drosha, DGCR8 and Dicer 1 indicated an impairment of miRNA biogenesis in response to AFB1. miR-34a was up-regulated significantly, down-regulating the expression of Wnt/β-catenin signaling pathway by target gene β-catenin. Anti-miR-34a can significantly relieved the down-regulated β-catenin and its downstream genes, c-myc and Cyclin D1, and the S-phase arrest in cell cycle induced by AFB1 can also be relieved. These results suggested that AFB1 might down-regulate Wnt/β-catenin signaling pathway in HepG2 cells by up-regulating miR-34a, which may involve in the mechanism of liver tumorigenesis.

Activation of oxidative stress and inflammatory factors could account for histopathological progression of aflatoxin-B1 induced hepatocarcinogenesis in rat.

Aflatoxin-B1 (AFB1) intoxication is known to develop hepatocellular carcinoma (HCC). However, pathogenesis and diagnosis of AFB1-induced HCC remain undefined. This article describes histopathological progression versus kinetics of the placental glutathione S-transferase (GST-pi) expression and profiles of the antioxidant enzymes, pro-inflammatory cytokines, and proliferative cell nuclear antigen (PCNA) in the liver from the rats administered with two doses of 1 mg AFB1/kg b.w. Histopathologically, hepatocytes necrosis could be observed after 4 weeks of the AFB1 treatment, which subsequently developed into the well-defined foci of altered hepatocytes (FAH) at 10 weeks post-treatment stage. This was consistent with an increasing trend of GST-pi immunostaining especially in the liver foci as a function of FAH progression and thus, suggesting that GST-pi over expression may serve as a marker for AFB1-induced hepatocarcinogenesis. The liver from AFB1-treated rats showed significantly enhanced level of reactive oxygen species coinciding with the declined immunostaining for superoxide dismutase-1, a committed enzyme of the antioxidant pathway, in the FAH regions and also with declined activity of the other antioxidant enzymes. Concordantly, the liver from the AFB1-treated rats showed over expression of pro-inflammatory cytokines; TNF-α & IL-1α and a cell proliferative marker PCNA. These findings present histological characterization of AFB1-induced HCC development and provide evidence for activation of oxidative stress-pro-inflammatory pathway during hepatocarcinogenesis induced by AFB1 toxicity.

Complete Protection against Aflatoxin B1-Induced Liver Cancer with a Triterpenoid: DNA Adduct Dosimetry, Molecular Signature, and Genotoxicity Threshold

In experimental animals and humans, aflatoxin B1 (AFB1) is a potent hepatic toxin and carcinogen. The synthetic oleanane triterpenoid 1-[2-cyano-3-,12-dioxooleana-1,9(11)-dien-28-oyl]imidazole (CDDO-Im), a powerful activator of Keap1-Nrf2 signaling, protects against AFB1-induced toxicity and preneoplastic lesion formation (GST-P-positive foci). This study assessed and mechanistically characterized the chemoprotective efficacy of CDDO-Im against AFB1-induced hepatocellular carcinoma (HCC). A lifetime cancer bioassay was undertaken in F344 rats dosed with AFB1 (200 μg/kg rat/day) for four weeks and receiving either vehicle or CDDO-Im (three times weekly), one week before and throughout the exposure period. Weekly, 24-hour urine samples were collected for analysis of AFB1 metabolites. In a subset of rats, livers were analyzed for GST-P foci. The comparative response of a toxicogenomic RNA expression signature for AFB1 was examined. CDDO-Im completely protected (0/20) against AFB1-induced liver cancer compared with a 96% incidence (22/23) observed in the AFB1 group. With CDDO-Im treatment, integrated level of urinary AFB1-N(7)-guanine was significantly reduced (66%) and aflatoxin-N-acetylcysteine, a detoxication product, was consistently elevated (300%) after the first AFB1 dose. In AFB1-treated rats, the hepatic burden of GST-P-positive foci increased substantially (0%-13.8%) over the four weeks, but was largely absent with CDDO-Im intervention. The toxicogenomic RNA expression signature characteristic of AFB1 was absent in the AFB1 + CDDO-Im-treated rats. The remarkable efficacy of CDDO-Im as an anticarcinogen is established even in the face of a significant aflatoxin adduct burden. Consequently, the absence of cancer requires a concept of a threshold for DNA damage for cancer development.

Hepatitis B Virus Genetic Variation and TP53 R249S Mutation in Patients with Hepatocellular Carcinoma in Thailand

Chronic hepatitis B virus (HBV) infection and dietary exposure to aflatoxin B1 (AFB1) are major risk factors for hepatocellular carcinoma (HCC). The aim of this study was to evaluate the role of HBV genetic variation and the R249S mutation of the p53 gene, a marker of AFB1-induced HCC, in Thai patients chronically infected with HBV. Sixty-five patients with and 89 patients without HCC were included. Viral mutations and R249S mutation were characterized by direct sequencing and restriction fragment length polymorphism (RFLP) in serum samples, respectively. The prevalences of T1753C/A/G and A1762T/G1764A mutations in the basal core promotor (BCP) region were significantly higher in the HCC group compared to the non-HCC group. R249S mutation was detected in 6.2% and 3.4% of the HCC and non-HCC groups, respectively, which was not significantly different. By multiple logistic regression analysis, the presence of A1762T/G1764A mutations was independently associated with the risk of HCC in Thai patients.

Anticarcinogenic activity of kalpaamruthaa, a modified Siddha preparation, against aflatoxin–B1-induced hepatocellular carcinoma in rats

To investigate the potential anticancer effect of kalpaamrutha (KA), a modified Siddha preparation, against aflatoxin–B1(AFB1)-induced hepatocellular carcinoma (HCC) in rats, KA (200 mg/kg body weight/day) was administered to AFB1-induced (2 mg/kg body weight i.p.) HCC rats, orally for 28 days. At the end of the experimental period, changes in body weight were recorded; the levels of alpha-fetoprotein and total protein was estimated in the serum; activities of marker enzymes were assayed in serum and total protein; DNA and RNA contents were estimated in liver tissue; activities of glycolytic enzymes, mitochondrial Krebs cycle enzymes, and respiratory chain enzymes were assayed in liver tissue of control and experimental rats. Further histopathological examination of the liver sections was carried out to support the anticancer effect of KA against AFB1-induced HCC. Administration of KA overall increase in glycolytic enzymes with a subsequent reduction in gluconeogenic enzymes, mitochondrial Krebs cycle enzymes, and respiratory chain enzymes was observed in HCC-induced rats. These altered enzyme activities were effectively counteracted by supplementation with KA and also prevented the body weight loss by enhancing the host energy metabolism. Histological studies supported the biochemical findings. The results of the present study reveal that the drug KA has potential anticancer effect against AFB1-induced HCC rats.

Potential Antioxidant Role of Tridham in Managing Oxidative Stress against Aflatoxin-B(1)-Induced Experimental Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is one of the most fatal cancers due to delayed diagnosis and lack of effective treatment options. Significant exposure to Aflatoxin B1 (AFB1), a potent hepatotoxic and hepatocarcinogenic mycotoxin, plays a major role in liver carcinogenesis through oxidative tissue damage and p53 mutation. The present study emphasizes the anticarcinogenic effect of Tridham (TD), a polyherbal traditional medicine, on AFB1-induced HCC in male Wistar rats. AFB1-administered HCC-bearing rats (Group II) showed increased levels of lipid peroxides (LPOs), thiobarbituric acid substances (TBARs), and protein carbonyls (PCOs) and decreased levels of enzymic and nonenzymic antioxidants when compared to control animals (Group I). Administration of TD orally (300 mg/kg body weight/day) for 45 days to HCC-bearing animals (Group III) significantly reduced the tissue damage accompanied by restoration of the levels of antioxidants. Histological observation confirmed the induction of tumour in Group II animals and complete regression of tumour in Group III animals. This study highlights the potent antioxidant properties of TD which contribute to its therapeutic effect in AFB1-induced HCC in rats.

Effect of probiotic fermented milk and chlorophyllin on gene expressions and genotoxicity during AFB 1-induced hepatocellular carcinoma

The aim of this study was to investigate the chemopreventive effect of probiotic fermented milk and chlorophyllin on aflatoxin B 1 (AFB 1) induced hepatocellular carcinoma. In vivo trials were conducted on 200 Wistar rats allocated to eight groups. Rats in the positive control group were given intraperitoneal injection of aflatoxin B 1 at 450 μg/kg body weight twice a week for 6 weeks. The rats were sacrificed and dissected at 25th week of the experiment, and comet assay was carried out in hepatic cells to assess the genotoxicity or DNA damage. The tumour incidence was decreased by approximately one-third than AFB 1 control group. The expression of c- myc bax, bcl- 2, cyclin D1, p53 and rasp- 21 genes was also studied. A significant (P < 0.05) reduction in DNA damage was observed in probiotic fermented milk with chlorophyllin group as compared to aflatoxin B 1 control group. The c- myc, bcl- 2, cyclin D1 and rasp- 21 level was found to be highest in AFB 1 control group as compared to the treatment group. The results advocate the enhanced protective potential of probiotic fermented milk and chlorophyllin against AFB 1-induced molecular alterations in hepatic cells during carcinogenesis.

Effect of Semecarpus anacardium nut milk extract on rat serum alpha-fetoprotein level in aflatoxin B 1-mediated hepatocellular carcinoma

An increased level of serum alpha-fetoprotein is observed in aflatoxin B 1 (AFB 1)-induced hepatocellular carcinoma (HC) in rats. Semecarpus anacardium nut milk extract (200 mg/kg, p.o., for 14 days) significantly reduced serum alpha-fetoprotein levels within the normal range, suggesting an antitumour activity against AFB 1-induced HC.

Expression of p53 gene in hepatocellular carcinomas induced by aflatoxin B1 with or without human hepatitis B virus in tree shrews

Using tree shrew as an animal model, our previous studies have demonstrated synergistic effects ofaflatoxin B₁(AFB₁) and human hepatitis B virus (HHBV) in the induction of hepatocellular carcinoma (HCC). In the present study, we have examined expression of p53 gene in HCCs induced by AFB₁ with or without HHBV infection in tree shrews. Avidin-biotin-peroxidase complex immunohistochemical method with human p53-CM₁ polyclonal antibody has been used to detect p53 expression in serial sections of paraffin-embedded liver and HCC tissues. Five out of 9 animals with HCCs (55.6%) induced by AFB₁ with HHBV infection and 2/3 animals with HCCs (66.7%) induced by AFB₁ alone expressed the p53 protein. Out of 18 HCCs examined, expression of p53 protein was observed in 9/10 moderately and poorly differ-entiated HCCs (90.0%). None of the well differentiated HCCs (0/8) expressed p53 (0%). Similarly, no p53 expression was observed in either non-tumorous or hyperplastic liver tissues or nodules. These results suggest that p53 expression associated with p53 mutation is a late event occurring probably during tumor progression in AFB₁ and HHBV induced hepa-tocarcinogenesis in the tree shrew. This report is the first example of an experimental animal model where combination of human HBV and AFB₁-induced HCCs demonstrate p53 expression.