Mycotoxins are conventional and novel risk biomarkers for hepatocellular carcinoma

Abstract

Hepatocellular carcinoma (HCC) is a common malignant disease with poor prognosis. To improve the clinical outcome, early diagnosis of HCC arising from nonviral agents and hepatitis virus is important. Among several etiological factors, mycotoxins defined as carcinogens by the International Agency for Research in Cancer might be one of the critical risk factors for nonviral HCC. Aflatoxin B1 is the most well-known carcinogenic mycotoxin for HCC, but the role of the other types of mycotoxin remains unclear. Several studies have reported that a chromatographic separation technique based on high-performance liquid chromatography can successfully detect the concentration of mycotoxins in plasma. In this article, we review recent studies of mycotoxin, and discuss its possible significance as a biomarker of HCC.

Core tip: Mycotoxins are one of the possible important carcinogens of hepatocellular carcinoma (HCC). Recently, a chromatographic separation technique based on high-performance liquid chromatography (HPLC) has been recognized as a useful method for the quantitative analyses of mycotoxins in the sera of individuals. HPLC-based analysis of mycotoxins in the clinical samples would provide some new epidemiological information about non-viral HCC.

COMMENTARY ON HOT TOPICS

Hepatocellular carcinoma (HCC) is a common malignancy, with a high prevalence worldwide[1,2]. The prognosis of HCC has remained poor, because many of the patients also have chronic liver diseases and are not suitable for radical surgical treatment[1-3]. Another obstacle to HCC treatment is that hepatoma cells display strong resistance against standard chemotherapeutic drugs[2,3]. To enable early diagnosis and treatment, understanding the etiological risk factors of HCC is desirable. Currently, approximately half of all HCC patients suffer from chronic hepatitis B and C virus (HBV and HCV) infection, and remaining cases are affected by various types of etiological factors including alcohol abuse, cigarette smoking, mycotoxins, obesity, and oral contraceptive drugs[4]. Intriguingly, growing evidence has suggested that the types of etiological risk factors for HCC might differ between geographic areas. For example, obesity-associated HCC has become one of the most important medical issues in developed countries[5], while food contamination with mycotoxins remains a critical risk factor for HCC in developing countries, including South and East Africa, India and China[6].

Mycotoxins as a major dietary risk factor for liver cancer

In recent years, the relationship between cancer risk and mycotoxin has been universally publicized by global surveillance of food contamination. Mycotoxins are secondary metabolites produced by fungi and are present in various types of stored grains, and most of them are resistant against cooking, freezing and digestion after intake of contaminated food. Several food-contaminating mycotoxins have been defined as harmful carcinogens by the International Agency for Research in Cancer (IARC) (Table 1), that is, deoxynivalenol/nivalenol, zearalenone, ochratoxin, fumonisins and aflatoxins. Of these, aflatoxin B1 (AFB1) is the most well-known bioaccumulative toxin involved in the development of HCC[7]. AFB1 is produced by the fungi Aspergillus flavus and Aspergillus parasiticus, and mainly contaminates improperly stored cereals and peanuts. When individuals are exposed to AFB1 for a long time, mono-oxygenases produce reactive epoxide in the liver, leading to formation of toxic derivatives with nucleic acids and proteins[8]. AFB1 has a strong mutagenic effect, and induces G to T transversion within codon 249 of the tumor suppressor p53 gene. Point mutation of p53 has been observed in approximately half of all patients with AFB1-associated HCC[9-11]. It has also been reported that geographical distribution of aflatoxin exposure and HBV infection overlap, leading to a synergistic effect on the genetic mutation of p53[10,11]. AFB1 is now regarded as the representative of orally ingested carcinogens, and has been classified as a Group 1 carcinogen by IARC (IARC 7th Annual Report on Carcinogens, 1987).

Table 1.

Classification of food mycotoxins as human carcinogens or potential human carcinogens

Group	Classification of food mycotoxins
1	Aflatoxin B1, B2, G1, G2
2A	-
2B	Aflatoxin M1, ochratoxin A, sterigmatocystin
3	Citrinin, patulin, luteoskyrin, cyclochlorotine, deoxynivalenol
4	-

Group 1: Carcinogenic to humans; Group 2A: Probably carcinogenic to humans; Group 2B: Possibly carcinogenic to humans; Group 3: Not classifiable as to its carcinogenicity to humans; Group 4: Probably not carcinogenic to humans (classified by the International Agency for Research in Cancer).

Ochratoxin A: A possible diagnostic marker of HCC?

Based on the clinical evidence of AFB1 in HCC, several researchers have turned their attention to the other type of mycotoxins. Ochratoxin A (OTA), which has been classified as a possible human carcinogen (Group 2B) by the IARC, is a secondary metabolite of Aspergillus and Penicillium fungi. OTA is widely spread in cereals such as barley, wheat, coffee and bread[12], and is well known for its possible contribution to nephritic diseases. Although the evidence is still open to debate, several studies have reported that OTA might be a causative agent of Balkan endemic nephropathy[13]. Moreover, OTA is increased to high levels in the plasma of patients with nephropathy in specific regions such as Tunisia[14], suggesting that OTA plays a critical role in the development of nephritic diseases.

Unfortunately, the role of OTA in hepatocarcinogenesis remains unclear. Although the results of relevant studies are controversial, several have suggested that the carcinogenic effect of OTA is due to increased hepatotoxicity and DNA damage. Ehrlich et al[15] have tested the genotoxic effect of OTA in human hepatoma HepG2 cells using both micronucleus and single-cell gel electrophoresis assays, and have found that it causes pronounced dose-dependent effects on DNA damage. Renzulli et al[16] have reported that OTA induces DNA damage through oxidative stress, and this could be prevented by rosmarinic acid, a natural phenolic compound contained in many Lamiaceae herbs. Bouaziz et al[17] have reported that OTA triggers a p53- and caspase-dependent mitochondrial apoptotic pathway in HepG2 cells. In contrast, El Golli Bennour et al[18] have reported that OTA does not induce significant reactive oxygen species generation in cultured HepG2 cells, but induces mitochondrial and caspase-dependent apoptotic cell death mediated by p53 transcription-independent activities. The aforementioned different and controversial studies suggest that etiological analysis of patients with HCC is indispensable for assessing the relationship between OTA and HCC.

Until recent decades, meta-analysis of etiological risk factors for carcinogenesis has been complicated because traditional methods for assessing toxin exposure were mainly performed by questionnaires or standard enzyme linked immunosorbent assay and these cannot assess minute quantities of environmental toxins. During the last decades, however, a chromatographic separation technique based on high-performance liquid chromatography (HPLC) has enabled us to assess the concentration of mycotoxins in clinical samples such as urine and plasma[19]. HPLC enables us to detect OTA at a low level of 0.005 ng/mL in plasma[20], which is often less than the mean level in healthy individuals; therefore, this analytical method is preferable for evaluation of the etiological significance of OTA. For example, Grosso et al[21] and Aslam et al[22] have examined the levels of OTA in the serum of bladder cancer patients by HPLC, and have reported that OTA is unlikely to be a risk factor for bladder cancer in Tunisia and Karachi. di Giuseppe et al[23] have examined serum OTA in patients in the Molise region in Italy, and have reported that the levels of OTA are significantly associated with C-reactive protein and cardiovascular risk score in men. These lines of evidence strongly suggest that examining mycotoxin such as AFB1 and OTA might be useful for investigating the molecular mechanism of HCC development.

Mycotoxin and hepatitis virus: Another mechanism of HCC development?

Of interest, basic studies have suggested that oncogenic role of mycotoxin might be significantly enhanced when the host cells are affected by hepatitis viruses. The most well-known story is the combination effect of hepatitis B virus X (HBX) protein with AFB1. Groisman et al[24] reported that expression of HBX impaired the repair of the DNA damage induced by AFB1 in cultured hepatoma cells, suggesting that the combination of HBX and AFB1 leads to enhanced DNA mutation. Madden et al[25] examined the rate of DNA mutation in HBX-transgenic mice, and found that the incidence of DNA mutations following AFB1 was increased to two fold in these animal models. More intriguingly, Li et al[26] implanted the HBX-transfected oval cells into nude mice with AFB1 treatment, and found that combination of HBx gene and treatment with AFB1 produces tumors in vivo. These lines of evidence strongly suggest that the long exposure to mycotoxin may cause unexpectedly enhanced tumorigenesis in the individuals with chronic hepatitis virus infection. Unfortunately, there have been no studies investigating the mechanism of combination of hepatitis viruses and mycotoxins. Further studies of the relationship between food contamination and hepatitis viruses might help to understand the molecular mechanism of hepatocarcinogenesis.

Summary

Recently, progress in the diagnosis and treatment of hepatitis virus infection has significantly improved the outcome of patients with HBV- and HCV-associated HCC. Early diagnosis of HCC arising in patients with nonviral disease, however, has been remained difficult. HPLC-based analysis of clinical samples might offer a useful tool for detecting minute concentrations of environmental toxins that have accumulated in the host. Although food contamination with mycotoxins has so far been recorded in specific geographic regions, it is conceivable that current advances in the transportation system could cause unexpected food contamination in a wide area. Therefore, to address whether mycotoxin would be a real causal agent for HCC, assessment of the level of mycotoxins, including OTA, in clinical samples would be of value.