Sulforaphane, a cancer chemopreventive agent, induces pathways associated with membrane biosynthesis in response to tissue damage by aflatoxin B1

https://doi.org/10.1016/j.taap.2014.11.004Get rights and content

Highlights

  • This study revealed sulforaphane (SF)-deregulated gene sets in aflatoxin B1 (AFB1)-treated rat livers.

  • SF redirects biochemical networks toward lipid biosynthesis in AFB1-dosed rats.

  • SF enhanced gene sets that would be expected to favor cell repair and regeneration.

Abstract

Aflatoxin B1 (AFB1) is one of the major risk factors for liver cancer globally. A recent study showed that sulforaphane (SF), a potent inducer of phase II enzymes that occurs naturally in widely consumed vegetables, effectively induces hepatic glutathione S-transferases (GSTs) and reduces levels of hepatic AFB1-DNA adducts in AFB1-exposed Sprague Dawley rats. The present study characterized the effects of SF pre-treatment on global gene expression in the livers of similarly treated male rats. Combined treatment with AFB1 and SF caused reprogramming of a network of genes involved in signal transduction and transcription. Changes in gene regulation were observable 4 h after AFB1 administration in SF-pretreated animals and may reflect regeneration of cells in the wake of AFB1-induced hepatotoxicity. At 24 h after AFB1 administration, significant induction of genes that play roles in cellular lipid metabolism and acetyl-CoA biosynthesis was detected in SF-pretreated AFB1-dosed rats. Induction of this group of genes may indicate a metabolic shift toward glycolysis and fatty acid synthesis to generate and maintain pools of intermediate molecules required for tissue repair, cell growth and compensatory hepatic cell proliferation. Collectively, gene expression data from this study provide insights into molecular mechanisms underlying the protective effects of SF against AFB1 hepatotoxicity and hepatocarcinogenicity, in addition to the chemopreventive activity of this compound as a GST inducer.

Introduction

Aflatoxin B1 (AFB1) is a known human carcinogen that significantly contributes to the burden of hepatocellular carcinoma (HCC) in many parts of the world, especially in areas with a warm and moist climate such as Asia and sub-Saharan Africa (Kensler et al., 2011). A critical mechanism responsible for the hepatotoxic and carcinogenic potential of AFB1 is based on the balance of its bioactivation and detoxification (Fig. 1). Several lines of evidence indicate that variation in the extent of glutathione (GSH) conjugation of the ultimate carcinogen, AFB1-8,9-epoxide, by glutathione S-transferases (GSTs) is an important detoxification pathway. Treatment of rats with oltipraz and 3H-1,2-dithiole-3-thione (D3T) leads to marked increases in the activity of liver GSTs, which resulted in reductions in both the extent of aflatoxin-DNA adduction and tumorigenesis (Kensler et al., 1987). The inducible A5 subunit of alpha-class GSTs in the rat has been identified as the GST isozyme that is primarily responsible for the enhanced detoxification of the AFB1-8,9-epoxide by chemopreventive agents (Hayes et al., 1991, Hayes et al., 1998). Modulation of GST activity is only one mechanism by which exogenous agents can influence aflatoxin carcinogenesis. This paper probes additional pathways.

Sulforaphane (SF), a potent isothiocyanate derivative found in broccoli and other cruciferous vegetables (Fahey et al., 1997), has received attention as a chemopreventive agent due to its ability to activate the transcription factor Nrf2 and induce phase II detoxification enzymes, including the GSTs (Fimognari and Hrelia, 2007, Myzak and Dashwood, 2006, Thimmulappa et al., 2002). Recent evidence illustrates protective effects of SF against AFB1-induced hepatotoxicity in rats attributable to increased GST expression. Treatment of rats with SF resulted in significant induction of hepatic total GST activity and a proportional reduction in the amounts of AFB1-N7-guanine (the principal DNA adduct of AFB1) formed in liver DNA (Fiala et al., 2011). Previous studies, however, have also identified additional chemopreventive mechanisms for SF that are independent of phase II enzyme induction. SF induces apoptosis in both in vitro (Fimognari et al., 2004, Karmakar et al., 2006) and in vivo models (Singh et al., 2004). SF-mediated cell cycle arrest has been reported in many previous studies, including induction of a dose-dependent growth arrest in prostate cancer cells by inhibiting the expression of cyclin D1 and DNA synthesis, along with a G1 cell cycle block (Chiao et al., 2002) and induction of G2/M accumulation and pre-metaphase arrest in bovine aortic endothelial (BAE) cells (Jackson et al., 2007). SF also exerts anti-inflammatory properties by inhibiting pro-inflammatory and pro-carcinogenic signaling factors such as IL-1β (Lin et al., 2008), COX-2 and TNF-α (Heiss et al., 2001). Understanding these phase II-independent pathways provides the rationale for the current project.

Cell regeneration and survival responses signal metabolic reprogramming that supports anabolic pathways required for tissue repair and growth (Ward and Thompson, 2012). The Keap1–Nrf2 complex, which is activated by SF, has been demonstrated to influence intermediary metabolism (Hayes and Dinkova-Kostova, 2014). This study aimed to assess the extent to which anabolic pathways modulated by SF provide protective mechanisms against AFB1 toxicity in vivo. The results revealed prominent reprogramming of gene sets involved in lipid synthesis in SF-pretreated rats, suggesting that SF facilitates regeneration of hepatic cells damaged by AFB1.

Section snippets

Chemicals

R,S-Sulforaphane (SF) was purchased from LKT Laboratories (St. Paul, MN). Aflatoxin B1 (AFB1) was purchased from Sigma Chemical (St. Louis, MO). RNAlater, RNeasy Mini Kit, and One-Step QuantiTech SYBR Green RT-PCR were obtained from QIAGEN (Valencia, CA). One-Cycle Target Labeling and Control Reagents complete kit (P/N 900493) and GeneChip® Rat Genome 230 2.0 arrays (Rat 230 2.0) were obtained from Affymetrix, Inc. (Santa Clara, CA). R-Phycoerythrinstreptavidin (SAPE) was purchased from

Gene expression analysis

The goal of this study was to compare the response of rat liver to aflatoxin alone, or aflatoxin in concert with SF. The transcriptional responses in the livers of AFB1-treated male rats induced by SF were investigated using a DNA microarray, an established tool to assess global gene expression changes. Groups of rats were pretreated with 0.7 mmol/kg SF by gavage at ages 30, 32, and 34 days, a dose previously shown to induce GSTs significantly and result in a substantial reduction of AFB1-N7

Discussion

AFB1 is one of the major risk factors for development of liver cancer, especially when it is present in concert with hepatitis B infection (Groopman and Kensler, 2005). Epidemiological investigations in human populations reveal an association of increased incidence of HCC with increasing dietary contamination by AFB1 (Wild and Gong, 2010). As the fungal species that produces AFB1 is ubiquitous, reduction of AFB1 exposure from ingestion may be problematic and impractical in certain areas of the

Conflict of interest

The authors declare no conflicts of interest in the support, conception, execution or publication of the work described herein.

Acknowledgments

This work was supported by National Institutes of Health grants ES016313, P30-ES002109, P01 ES006052, P30 ES003819, and P30 CA006973. The authors thank Drs. Stephen Slocum and Daam Settachan for critically reading the manuscript.

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