The case for aflatoxins in the causal chain of gallbladder cancer

Abstract

Chronic aflatoxin exposure has long been related to hepatocellular carcinoma (HCC). Recently, its association with gallbladder cancer (GBC) was postulated. Here we present the data supporting this hypothesis in Chile, the country with the highest GBC mortality worldwide with age-standardized mortality rates (ASMR) of 10.3 in women and 5.04 in men.

The highest GBC rates occur in Southern Chile (ASMR= 18), characterized by: high Amerindian ancestry, associated with high bile acid synthesis and gallstones; high poverty and high cereal agriculture, both associated with aflatoxin exposure. Aflatoxins have been detected in imported and locally grown foods items. We estimated population dietary exposure ranging from 0.25 to 35.0 ng/kg-body weight/day. The only report on human exposure in Chile found significantly more aflatoxin biomarkers in GBC than in controls (Odds Ratio= 13.0).

The hypothesis of aflatoxin-GBC causal link in the Chilean population is supported by: genetically-determined rapid cholesterol excretion and high gallstones prevalence (49.4%); low prevalence of HCC (ASMR=4.99) and low HBV infection (0.15%) the main co-factor of aflatoxins in HCC risk.

If the association between aflatoxins and GBC were confirmed, public health interventions based on food regulation could have a substantial public health impact.

Introduction

A previous report hypothesized that excretion of excess of cholesterol from the liver into the gallbladder can lead to the simultaneous pumping of a food carcinogen into the bile. This carcinogen would then be stored and concentrated in the gallbladder, increasing the risk of gallbladder cancer (GBC) (1). One xenobiotic of particular interest is aflatoxin. Here we explore the data that supports the hypothesis that chronic exposure to aflatoxin may be a causal factor for GBC. Chile is the ideal place to study this hypothesis given its high burden of GBC in geographic defined areas.

Aflatoxins

Aflatoxins (B1, B2, G1 and G2) are secondary metabolites of Aspergillus flavus and Aspergillus parasiticus fungi that naturally contaminate food, including a wide range of products like cereals, oilseeds, spices, tree nuts, milk, meat and dried fruit (2). Agricultural products grown between latitudes 40° North and 40° South are at highest risk of becoming contaminated with aflatoxins and risk of consumption of contaminated products is particularly high in developing countries, associated with poverty, shortage and no strict food regulations (3,4,5). However, because of globalization no Region of the world is free from these toxins (6).

Aflatoxins are potent hepatotoxins and carcinogens, being aflatoxin B1 (AFB1) the most prevalent and toxic (7). The International Agency for Research on Cancer (IARC) classifies aflatoxins as carcinogenic to humans (Group 1) based on cohort studies which found increased risk for hepatocellular carcinoma (HCC) in individuals exposed to aflatoxins (relative risk=1.6–16.1), as well as several case-series and case–control studies. Further, carcinogenicity of aflatoxins has been shown in several experimental animal and mechanistic studies (6). The mutagenesis process is the result of the metabolism of aflatoxin by cytochrome P450s (CYPs) to the reactive 8,9-epoxide form, a genotoxic metabolite that can bind to essential proteins and react with DNA in the N7 position of guanines, forming DNA adducts. AFB1 adducts can result in GC to TA transversions of the tumor suppressor TP53 gene, which can lead to cancer (8). According to the IARC TP53 database, 39% of the HCC is associated to this transvertion followed by transitions GC to AT (9). In areas where aflatoxin exposure is high, up to 50% of HCC tumors have a specific point mutation in codon 249 of this gene (6).

Susceptibility to aflatoxin hepatotoxicity is associated with the expression level of CYP enzymes in the liver (10). Cofactors for aflatoxin-related carcinogenesis include liver infection and inflammation (11), nutritional factors, younger age and testosterone concentration (3); but aflatoxin’s most potent cofactor is hepatitis B virus (HBV) (6, 12, 13). HBV may increase aflatoxin metabolism (6) through induction of the CYPs, increasing the 8,9-epoxide in the liver. Also, chronic HBV infection leads to necrosis and regeneration of hepatocytes, which may predispose to TP53 mutation when exposed to aflatoxin (13).

Given the genotoxic properties of the metabolized aflatoxins, we hypothesize that these toxins are a causal factor for other digestive cancers, if specific co-factors were present. In particular, we aim to review the basis and evidence that chronic exposure to aflatoxins is a causal factor for GBC in the presence of high gallstone prevalence.

Gallbladder cancer

GBC is the most common malignancy of the biliary tract, with striking geographic variation worldwide, suggesting a strong role of environmental factors. The highest rates of GBC are found in Latin America and Asia, with intermediate rates in Eastern and Central Europe, and very low rates in the United States and most Western and Mediterranean European countries (14).

Known risk factors for GBC include gallstones (15), obesity, parity (16), increased endogenous and exogenous estrogen levels (17–19) and poverty (20). GBC risk has also been positively associated with total carbohydrate and calorie intake (21) and red chili pepper consumption (22,23). The association with red chili pepper consumption is in apparent contradiction with the anti-carcinogenic effect of capsaicin, its active compound. This paradox could potentially be explained by contamination of chili pepper with carcinogens (24).

GBC is more frequent in populations with a high prevalence of gallstones, such as the Amerindians of North, Central and South America (Table 1) (28, 32), and the risk is particularly high among Amerindian women of low socioeconomic status (20, 33). In Chile, it has been shown that Amerindians are especially susceptible to GBC due to the presence of lithogenic cholesterol genes (26) with an associated increase in bile acid synthesis (34).

Table 1.

Prevalence of gallstones in selected populations.

Selected populations (Reference)	Gallstones (%)
	Women	Men
North- Amerindians (25)	64.1	29.5
South- Amerindians (26)	49.4	12.6
Chileans (26)	37.4	14.5
Peruvians (27)	16.1	10.7
Bolivians (28)	15.7	7.5
White- American (25)	16.6	8.6
Black- American (25)	13.9	5.3
Brazilian (28)	6	2
Chinese (29)	3.7	6.9
Sub-Saharan Black Africans (25,30,31)	2.9–5.1	5.1

GBC incidence and mortality rates in Chile are among the highest in the world with age-standardized mortality rates (ASMR) of 10.3 in women and 5.04 in men (35). In 2006, a GBC prevention program was implemented in the Country which was based in preventive cholecystectomy for people aged 35 to 49 years carrying gallstones, resulting in thousands of patients being cholecystectomized annually (36). But health care resources have been insufficient to cover the target population.

GBC is more frequent in the Southern Regions of Chile (VII to X) (20, 33, 37), where the proportion of Amerindian population (38) and of poverty (39) are the highest (Figure 1). Since aflatoxins and GBC are both associated with poverty and the latter with Amerindian origin (4, 5, 20), aflatoxins may be the environmental factor in the gene-environment causal chain to GBC.

Figure 1.

GBC mortality, poverty and Amerindian in the Chilean Regions from North to South.

Hypothesis

Chronic exposure to aflatoxins will preferentially increase risk of GBC rather than HCC in populations with 1) genetic predisposition toward rapid cholesterol excretion into the gallbladder, 2) high prevalence of gallstones, and 3) low prevalence of HBV infection.

Evaluation of the hypothesis

Experimental and epidemiological data

Biliary excretion is the major excretory route of metabolized aflatoxin, which can reach high concentrations in the bile (40,41); thus, the gallbladder is a repository of these toxins. In experimental animals (pigs and dogs), a single oral dose of aflatoxin causes inflammation, edema and hemorrhage of the gallbladder; subsequent daily doses cause biliary proliferation (42). Sieber et al. (43) reported gallbladder and bile duct tumors developing in nonhuman primates 10 to 12 years after receiving AFB1 orally over a course of 117–136 weeks (515.49 to 1354.24 mg total AFB1).

The first and only report on human exposure, a case control study by Nogueira et al. (23) in Chile, found significantly more circulating aflatoxin-albumin adducts in GBC patients compared to gallstone patients (OR= 6.8; 95% CI, 1.9 to 29.0) or population controls (OR= 13.0; 95% CI, 3.0 to 52.5). In this study, 35% of participants have detectable levels of aflatoxin-albumin adducts, ranging from 0.3 to 238.45 pg/mg (23).

As in HCC, somatic TP53 mutations associated with GBC are common (44–47). According to the IARC TP53 database, most of mutation types associated to GBC are GC to AT transitions, having GC to TA transvertions in 8.18% of the cases (9). Mutations in TP53 related to GBC have shown geographic and ethnic variation suggesting both exogenous and endogenous causation (47). In Chile, TP53 mutations associated to GBC showed GC to TA transvertions in 6% of the cases (44); similar of what is shown in the IARC database.

Sources of exposure to aflatoxins

Food

Information of aflatoxin in food in Chile is scarce. In 2009, the Chilean government established aflatoxin regulation and in 2015 established limits of 10 ppb for total aflatoxin and 0.5 ppb for aflatoxin M1, a metabolite of AFB1 excreted through milk (48). Aflatoxin regulation control has consisted in a non-systematic sampling of approximately 40 food items per year, covering a few Regions, resulting in aflatoxin detection in some food items tested, mainly imported peanuts and spices but also in locally grown foods like corn and powdered chili (Table 2).

Table 2.

Aflatoxins in food and estimated mean dietary exposure of the Chilean population, based on the Global Environment Monitoring System consumption Cluster diets (56).

Food commodity (Reference)	Positive samples	Aflatoxins levels (ppb)	Year of sampling	Origin	C05 consumption g/day/person	Aflatoxins mean level (ppb)	Mean exposure (ng/kg-bw/day)c
Corn (49)	1/9 (11)	5	1990	National	Cereals: 358.4	5	29.87
Peanut (49)	1/9 (11)	60	1989	Imported	Treenuts: 15.9	7.32	1.94
Japanesse peanut (50)	1/3 (33)	33.3	2009	Imported
Almondsa	5/5 (100)	2.1 −3.1	2013	Imported
Peanuta	1/11 (9)	0.2	2013	National
Nutmeg (51)	5/5 (100)	14.5 – 35.8	2010	Imported	Spices: 4.4	3.38	0.25
Red chili (51)	3/3 (100)	9.9 – 10.9	2010	Imported
Nutmeg (52)	5/5 (100)	173.3	2011	Imported
Curry (52)	1/5 (20)	23	2011	Imported
Red chili (53)	1/1 (100)	4.9	2011	National
White pepper (54)	3/3 (100)	2.5– 4.7	2012	Imported
Black pepper(54)	2/4 (50)	2.6– 3.3	2012	Imported
Paprika (54)	3/4 (75)	1– 1.3	2012	Imported
Merquena	5/15 (33)	0.2–0.9	2013	National
Milk	N.Sb	N.Sb			Milk: 174.9	1	2.92
						Total	34.97

^aPublic Health Institute of Chile 2013, unpublished data.

^bNo sampled in Chile. It was assumed a mean concentration of 1 ppb.

^cDietary exposure (mean concentration level in food x daily amount consumed/body weight) measured in ng per kg of body weight per day, calculated with an average of 60 kg of body weight.

We estimated the Chilean dietary exposure to aflatoxins (according to 55) based on: i) Chilean (and other Latin-American countries) typical diet according to the WHO Global Environment Monitoring System (GEMS) (56) that estimates a daily consumption per person of 15.9 g of nuts, 4.4 g of spices, 358.4 g of cereals and 175 g of milk and; ii) aflatoxin mean levels of the food items measured in Chile (Table 2). We estimated that Chileans have an intermediate level of exposure ranging from 0.25 to 34.97 ng/kg-body weight (bw)/day, with cereal as the main contributor (Table 2). Based on these estimates, Chileans would consume higher amounts of aflatoxins than Europeans (0.93–2.45 ng/kg-bw/day) or residents of the United States (2.7 ng/kg-bw/day) but lower than residents in Africa (0.3–180 ng/kg-bw/day) or Asia (0.3–53 ng/kg-bw/day) (57).

Occupational

While the main source of aflatoxin exposure is the ingestion of contaminated food, inhalation could be an important route of exposure for agricultural workers during crop harvest and storage (58–60) and cereal handlers like bakers and mill workers (61). Occupational exposure to aflatoxin has been associated with HCC (61, 62) and other digestive cancers, such as esophageal cancer in Iran (63) and gallbladder and extrahepatic bile duct cancers in Denmark (64). In Chile, cereal production (mainly corn, wheat and rice) is concentrated in the Central and Southern Regions (between Regions IV and X) (65), coinciding with the high risk areas of GBC (Figure 1). Thus, occupational exposure to aflatoxin may also contribute to GBC risk among the agricultural workers of Southern Chile.

Plausibility of a causal role of aflatoxins in GBC

If aflatoxin exposure were common in Chile, however, it begs the question why Chile has relatively low rates of HCC (Table 3). One reason is the very low rate of HBV infection (0.15%) (70), the most potent co-factor for aflatoxin in HCC. Additionally, some have proposed that the metabolic disorder behind gallstones somehow protects the liver from xenobiotics (1). Individuals who synthetize and rapidly excrete excess of cholesterol into bile are prone to develop gallstones (34), and since xenobiotics are excreted together with cholesterol (71), the time that xenobiotics remain in the liver is reduced in rapid cholesterol excretors. By the same token, xenobiotics accumulate in the gallbladder. In addition, gallstones cause local mucosal irritation and chronic inflammation (72) increasing aflatoxin toxicity in the gallbladder. Another co-factor acting in Chile is gallbladder chronic infection, in particular by Salmonella typhi. Bacterial chronic infection may contribute to malignant transformation through the degradation of bile constituents, chronic inflammation or alteration of tumor suppressor genes (28, 73). Chile suffered a high outbreak of typhoid fever from 1976 to 1986 (73), leaving behind a large reservoir of chronic carriers (74) who are thought to be at increased risk of GBC (Koshiol J personal communication).

Table 3.

Hepatocellular carcinoma (HCC) and gallbladder cancer (GBC) incidence, percent of population infected with hepatitis B (HBV) infection and aflatoxin mean exposure in selected countries.

	HCC (35)	GBC (35)	% HBV (66)	Aflatoxin exposure (ng/kg-bw/day) (55)
Chile	4.66	9.74	0.5	0.25–35b
Bolivia	3.65	8.07	2 (0.5 to 16.7a)	0.25–35b
Brazil	4.56	1.88	2	0.23– 50
Malaysia	5.99	1.01	5	15–140
Peru	6.39	4.01	14 (<1 to 14a)	0.25–35b
China	22.27	2.83	12	17–37
The Gambia	25.83	0	14	4–115

^aThese countries shared areas with low prevalence in major cities and very high prevalence in rural zones near Amazon basin (67–69).

^bBolivia and Peru assuming similar exposure than Chile.

According to our hypothesis, the rapid excretion of xenobiotics protects the liver from the hepatocarcinogenic toxin. The fact that HCC mortality has been historically low in Chile is consistent with this hypothesis. Intriguingly, an upward trend in HCC mortality has been reported over the last 15 years, concurrent with the stabilization of GBC mortality (37) (Figure 2). The increase in HCC could be the result of the obesity epidemic (75) while the drop of GBC could be the result of the systematic increase in the cholecystectomy rate observed over the last 20 years. Another explanation of the reverse trend of these two cancers is that cholecystectomy increased risk of HCC, which has been reported in several cohort studies (76–78).

Figure 2.

GBC and HCC mortality in Chile 1997– 2011.

Thus, the lack of HBV for liver carcinogenesis and the potential for genetic susceptibility through dominant lithogenic genes, widely distributed in populations with South Amerindian ancestors (26), may explain how aflatoxins could contribute to high rates of GBC without causing high rates of HCC.

Discussion

Chronic diseases like cancer are multifactorial and depend on the interaction of genetics with environmental and other cofactors (e.g., chronic infections). Levels of exposure to the carcinogen are also important. High levels of aflatoxin biomarkers (> 250 pg/mg aflatoxin-albumin adduct) have been found in regions with high risk of HCC such as The Gambia (79) and China (80), which also have high rates of HBV infection (Table 3). Other cofactors for aflatoxin may include Hepatitis C virus in Taiwan (81) and malaria, kwashiorkor and HIV/AIDS in Africa (5); all of which are very low in Chile. High HBV rates and low genetic predisposition to gallstones (Table 1) could explain the low GBC incidence rates in Africa (Table 3).

In countries with intermediate exposure to aflatoxins like Chile, Malaysia (82, 83) and Brazil (84), HCC incidence will vary according to the HBV rate (Table 3). The highest GBC incidences in the world are in Chile and Bolivia (Table 3), which share the same GEMS diet with possibly similar aflatoxin exposure, Amerindian miscegenation prone to lithogenic genes and gallstones (Table 1) and low HBV rates (Table 3). HBV is low to intermediate in most Latin American countries, but is highly endemic in the Amazon Basin (85), shared by Peru, Bolivia and Brazil; up to 14% of some Amazon rural groups are hepatitis B virus surface antigen (HBsAg) seropositive (67). This may explain why Peru, with the same GEMS diet and Amerindian miscegenation as Chile, presents higher HCC rates and intermediate GBC rates (Table 3).

Conclusion

Epidemiological, experimental and occupational data supports the hypothesis that aflatoxins are carcinogens for the gallbladder. Low population rates of HBV infection, genetic predisposition to rapid cholesterol synthesis and excretion, high prevalence of gallstones and chronic bacterial infection may help explain the aflatoxin-GBC association in Chile. The characterization of aflatoxin exposure in Chile through biomarkers studies in the population and measurement of aflatoxin levels in incriminated food-items is urgently needed.

If aflatoxins were a causal factor for GBC, public health intervention should move from its current emphasis on secondary prevention through cholecystectomy to invest in primary prevention through food safety regulation, which would likely have a broader and more cost-effective impact.