Association of aflatoxin with gallbladder cancer in a case-control study nested within a Chinese cohort.

We evaluated whether aflatoxin B1 (AFB1 ) exposure was associated with later risk of developing gallbladder cancer (GBC). We measured AFB1 -lysine albumin adducts in baseline samples from the Shanghai Cohort Study of 18 244 men aged 45 to 64 years (recruited 1986-1989). We included 84 GBC cases with sufficient serum and 168 controls matched on age at sample collection, date of blood draw and residence. We calculated adjusted odds ratios (ORs) and 95% confidence intervals (95% CIs) for detectable vs non-detectable AFB1 -lysine albumin adducts and gallbladder cancer. AFB1 -lysine albumin adducts were detected in 50.0% of GBC cases, and risk of GBC was twice as high in those with detectable vs undetectable levels (OR = 2.0, 95% CI = 1.0-3.9). ORs ranged from 1.8 (95% CI = 0.75-4.3) for 0.5 to <1.75 pg/mg vs undetectable adduct levels to 2.2 (95% CI = 0.91-5.6) for >3.36 pg/mg vs undetectable, suggesting a dose-response (Ptrend = .05). When restricted to cases diagnosed before the median time to diagnosis after blood draw (18.4 years), results were similar (OR = 2.2, 95% CI = 0.80-5.8) to those for the entire follow-up duration. The OR was 9.4 (95% CI = 1.7-51.1) for individuals with detectable AFB1 -lysine albumin adducts and self-reported gallstones compared to individuals with neither. Participants with detectable AFB1 -lysine albumin adducts at baseline had increased risk of developing GBC, replicating the previously observed association between AFB1 exposure and providing the first evidence of temporality.

Association of Aflatoxin and Gallbladder Cancer.

BACKGROUND & AIMS: Aflatoxin, which causes hepatocellular carcinoma, may also cause gallbladder cancer. We investigated whether patients with gallbladder cancer have higher exposure to aflatoxin than patients with gallstones. METHODS: We measured aflatoxin B1 (AFB1)-lysine adducts in plasma samples from the Shanghai Biliary Tract Cancer case-control study, conducted from 1997 through 2001. We calculated age- and sex-adjusted odds ratios (ORs) and 95% confidence intervals (CIs) and the population-attributable fraction for 209 patients with gallbladder cancer and gallstones vs 250 patients with gallstones without cancer (controls). In 54 patients with gallbladder cancer, tumor tissue was examined for the R249S mutation in TP53, associated with aflatoxin exposure, through targeted sequencing. RESULTS: The AFB1-lysine adduct was detected in 67 (32%) of 209 patients with gallbladder cancer and 37 (15%) of the 250 controls (χ2 P < .0001), almost threefold more patients with gallbladder cancer than controls (OR, 2.71; 95% CI, 1.70-4.33). Among participants with detectable levels of AFB1-lysine, the median level of AFB1-lysine was 5.4 pg/mg in those with gallbladder cancer, compared with 1.2 pg/mg in controls. For patients in the fourth quartile of AFB1-lysine level vs the first quartile, the OR for gallbladder cancer was 7.61 (95% CI, 2.01-28.84). None of the 54 gallbladder tumors sequenced were found to have the R249S mutation in TP53. The population-attributable fraction for cancer related to aflatoxin was 20% (95% CI, 15%-25%). CONCLUSIONS: In a case-control study of patients with gallbladder cancer and gallstones vs patients with gallstones without cancer, we associated exposure to aflatoxin (based on plasma level of AFB1-lysine) with gallbladder cancer. Gallbladder cancer does not appear associate with the R249S mutation in TP53. If aflatoxin is a cause of gallbladder cancer, it may have accounted for up to 20% of the gallbladder cancers in Shanghai, China, during the study period, and could account for an even higher proportion in high-risk areas. If our findings are verified, reducing aflatoxin exposure might reduce the incidence of gallbladder cancer.

Exploring the potential carcinogenic role of arsenic in gallbladder cancer.

Gallbladder cancer (GBC) is an aggressive malignancy, associated with dismal outcomes. Although several risk factors including age, sex, and gallstones have been postulated, epidemiologic determinants of the disease remain largely uncovered. Moreover, the implication of environmental toxicants as possible risk factors is increasingly suspected. Arsenic (As), an established human carcinogen, is a natural contaminant of groundwater and has a geographic distribution similar to GBC incidence. This, combined with As metabolites being partially excreted in bile, raised the hypothesis that As may represent a carcinogenic hazard for the gallbladder. We conducted an analysis of the association between As concentration in groundwater and incidence rates of GBC worldwide in 52 countries. The USA, India, and Taiwan were selected on the basis of availability and quality of data for further investigation at a county-level. Relationships between As levels and GBC incidence were assessed using multivariable linear regression analyses. Analyses revealed significant associations between high As concentrations in groundwater and increased GBC incidences. Among women, correlations were observed worldwide (Spearman = 0.31, P = 0.028), in Taiwan (Spearman = 0.57, P = 0.005) and in India (R = 0.23, P = 0.006). In men, a correlation was observed in India (R = 0.26, P = 0.009) and a modest correlation was identified in the USA (Spearman = 0.14, P = 0.026). These results provide some support to the hypothesis of an association between high exposures to As-contaminated water on GBC, which appeared more prominent in women. Further observational and molecular studies, conducted at the individual level, are required to confirm this association and decipher its nature.

Tumors induced in mice by N-methyl-N-formylhydrazine of the false morel Gyromitra esculenta.

Continuous administration of 0.0078% N-methyl-N-formylhydrazine (MFH) in drinking water to 6-week-old outbred Swiss mice for life produced tumors of the liver, lung, gallbladder, and bile duct. The incidences of tumors in these four tissues were 33, 50, 9, and 7%, whereas in the untreated controls they were 1, 18, 0, and 0%, respectively. The higher dose (0.0156% MFH) given under identical conditions had no tumorigenic effect, since it proved too toxic for the animals. Histopathologically, the lesions were classified as benign hepatomas, liver cell carcinomas, adenomas and adenocarcinomas of the lungs, adenomas of the gallbladder, cholangiomas, and cholangiocarcinomas. Since the edible false morel Gyromitra esculenta contains a high amount of MFH, the human population should be dissuaded from consumption of this dangerous mushroom.

A potent pancreatic carcinogen in Syrian hamsters: N-nitrosobis(2-oxopropyl)amine.

N-Nitrosobis(2-oxopropyl)amine (BOP), a further postulated beta-metabolite of di-n-propylnitrosamine, induced a high incidence of pancreatic duct adenomas and adenocarcinomas as early as 13 weeks in Syrian hamsters receiving weekly sc injections for life and a few pancreatic adenomas, after 28 weeks, in those given a single sc dose. Compared to related compounds, N-nitrosobis(2-hydroxypropyl)amine and N-nitrosobis(2-acetoxy-propyl)amine which are also pancreatic carcinogens, BOP induced only a few neoplasms of the lung, liver, and kidney and none in the nasal cavity, larynx, and trachea. The results therefore indicate progress in developing a more specific model for pancreatic carcinogenesis studies.

Effects of phenobarbital and secondary bile acids on liver, gallbladder, and pancreas carcinogenesis initiated by N-nitrosobis (2-hydroxypropyl)amine in hamsters.

The effects of dietary administration of phenobarbital [(PB) CAS: 50-06-6] or the secondary bile acids, deoxycholic acid [(DCA) CAS: 83-44-3] and lithocholic acid [(LCA) CAS: 434-13-9], on tumorigenesis in the liver, gallbladder, and pancreas were investigated in male Syrian golden hamsters after carcinogenic initiation by N-nitrosobis(2-hydroxypropyl)amine [(BHP) CAS: 53609-64-6]. BHP [500 mg/kg (body wt)] was injected sc once weekly for 5 weeks. The animals were then maintained on a basal diet or a diet containing either 0.05% PB, 0.1% DCA, 0.5% DCA, or 0.5% LCA for 30 weeks. DCA enhanced the development of cholangiocarcinomas without influencing that of hepatocellular lesions. PB promoted the induction of hepatocellular carcinomas but not that of cholangiocarcinomas. LCA was without effect on the induction of either hepatocellular carcinomas or cholangiocarcinomas. DCA at a dose of 0.5% enhanced the induction of polyps in the gallbladder. Both DCA, at a dose of 0.1%, and LCA significantly enhanced the induction of pancreas carcinomas. PB had no effect on the induction of polyps in the gallbladder or of pancreas carcinomas. These data document that different tumors may be differentially promoted following initiation with a common carcinogen.

Effect of beta-oxidized nitrosamines on syrian hamsters. III. 2,2'-Dihydroxydi-n-propylnitrosamine.

2, 2-Dihydroxy-di-n-propylnitrosamine (DHPN), an assumed metabolite of di-n-propylnitrosamine (DPN), injected subcutaneously once weekly for life, was carcinogenic in Syrian hamsters. The main target organs were the respiratory tract, pancreas, liver, and kidneys. In the respiratory system the most affected segments were the nasal cavities and the lungs. Adenomas and adenocarcinomas, mostly of ductal origin, were induced in the pancreas. Liver neoplasms were hemangloendotheliomas, angiosarcomas, hepatocellular adenomas, cholangiomas, and cholangiocarcinomas. Kidney neoplasms were adenomas and adenocarcinomas. The morphology of the induced neoplasms was described, as well as the effects of DHPN, compared to those another possible metabolite of DPN, 2-hydroxypropyl-n-propylnitrosamine (2-HPPN), which is formed in vivo with only 1 aliphatic chain degraded via theta-oxidation.

N-nitroso-bis(2-acetoxypropyl)amine as a further pancreatic carcinogen in Syrian golden hamsters.

N-Nitroso-bis(2-acetoxypropyl)amine, a possible beta metabolite of N-nitroso-di-n-propylamine, was shown to be a potent carcinogen in the Syrian golden hamster. After a single s.c. treatment, the pancreas was the most affected organ, followed by the liver, respiratory tract, and kidneys. However, repeated application resulted in a higher incidence of neoplasms of the respiratory tract than of the pancreas and kidneys. The effect of N-nitroso-bis(2-acetoxypropyl)amine on toxicity, target tissues, and carcinogenicity was similar to that of N-nitroso-bis(2-hydroxypropyl)amine. The assumption that these two compounds may have similar metabolic pathways was confirmed; N-nitroso-bis(2-acetoxypropyl)amine was readily deesterified to N-nitroso-bis(2-hydroxypropyl)amine in vivo and in vitro.

Orotic acid enhancement of preneoplastic and neoplastic lesions induced in the pancreas and liver of hamsters by N-nitroso(2-hydroxypropyl) (2-oxopropyl)amine.

The effect of dietary orotic acid (OA) in liver-pancreas carcinogenesis induced in female Syrian hamsters by N-Nitroso(2-hydroxypropyl) (2-oxopropyl)amine (HPOP) was evaluated. All animals infused with the carcinogen received the same doses. Results of the control group which received no OA or carcinogen were compared with the results of: (a) hamsters treated with HPOP and fed a regular 20% protein synthetic diet (group 1); (b) hamsters fed the OA diet for a brief time period during initiation with the carcinogen (group 2); and (c) hamsters in which OA was administered after carcinogen infusion for life (group 3). All animals of the control group were normal at autopsy, while those in group 1 (HPOP alone) revealed the spectrum of lesions accepted as classical in the multistep hyperplasia-dysplasia-carcinoma in situ (CIS) sequence of carcinogenesis. Results of group 2, in light of group 1, revealed an increased incidence of the following lesions in the common pancreatic duct: dilatation, 2.5 times; flat and papillary hyperplasia, 2 times; and dysplasia (atypical hyperplasia), 12 times. No significant increase of CIS and invasive cancer in the body and tail of the pancreas was observed; in addition, the incidence, nature, and location of pancreatic adenocarcinomas were not affected. Yet, the effect of OA administered after carcinogen infusion (group 3) when compared to group 1 seemed to enhance a further increase in the incidence of practically all lesions throughout the pancreas. An obvious overall step-up incidence along the multistep hyperplasia-dysplasia-CIS-invasive cancer process in the pancreas was observed. The increase in incidence of flat, papillary, and atypia of the epithelium of the common pancreatic duct in group 3 was mild compared to that found in the same duct of group 2, but the increase in incidence of these same three lesions when found in the main ducts was marked: flat hyperplasia, 3-fold; papillary hyperplasia, 2.5-fold; atypical hyperplasia, 3-fold. The increase in incidence of CIS in this group was 5-fold and papillary adenocarcinomas, 3-fold, when compared to 5% found in groups 1 and 2. Hepatic malignancies (cholangiocarcinomas) occurred in 6% of the cases in group 3 compared to none in group 2; the incidence of malignancy in the gallbladder was the same in groups 2 and 3 but three times greater than that in group 1.(ABSTRACT TRUNCATED AT 400 WORDS)

Inhibition of streptozotocin-induced islet cell tumors and N-nitrosobis(2-oxopropyl)amine-induced pancreatic exocrine tumors in Syrian hamsters by exogenous insulin.

The effects of streptozotocin (SZ) and N-nitrosobis(2-oxopropyl)amine (BOP), separately or in combination, on the pancreas, common duct, and gallbladder, all target tissues of BOP, were studied in Syrian golden hamsters. Groups of hamsters were treated with either a single dose (20 mg/kg body weight) of BOP (BOP group), or a single i.p. dose (50 mg/kg body weight) of SZ and 14 days later with a single s.c. injection of the same dose of BOP (SZ + BOP group). Another group of animals was treated similarly with BOP and SZ except that they received twice daily injections of insulin, beginning 1 day after SZ administration and for the duration of the experiment (52 weeks) (SZ + insulin + BOP group). The control group consisted of hamsters treated with a single dose of BOP and daily doses of insulin (insulin + BOP group). Hamsters treated with SZ recovered spontaneously from their diabetes, although the mortality was high (86%). BOP treatment inhibited the diabetogenic effects of SZ in both SZ + BOP and SZ + insulin + BOP groups and reduced the mortality to 43 and 74%, respectively. SZ pretreatment inhibited the incidence of BOP-induced pancreatic ductal/ductular cell carcinomas in the SZ + BOP group (P less than 0.01); this protective effect of SZ on carcinoma development was potentiated by additional treatment with insulin (SZ + insulin + BOP group, P less than 0.001). Although the frequency of BOP-induced tumors in the gallbladder (all polyps) was not altered by either SZ or insulin, the frequency of the common duct polyps was significantly lower in the SZ + insulin + BOP group than in the BOP group (P less than 0.005). Hamsters in the SZ, SZ + BOP, and SZ + insulin + BOP groups developed islet cell adenomas (insulomas). However, the SZ + insulin + BOP group had significantly fewer insulomas than in the SZ + BOP group (P less than 0.0005). The overall data confirm the inhibitory effect of SZ on BOP-induced pancreatic cancer and suggest that this effect is related to the diabetic condition of hamsters rather than insulin deficiency and that intact islets appear to be prerequisite for exocrine pancreatic cancer induction by BOP. On the other hand, the inhibitory action of insulin on insuloma induction by SZ and on ductal/ductular cancer induction by BOP seems to be related to the suppressive effect of this hormone on beta-cell and ductal/ductular cell replication, respectively.

The case for aflatoxins in the causal chain of gallbladder cancer.

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=4.9) 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.

Ochratoxin A Contamination of Red Chili Peppers from Chile, Bolivia and Peru, Countries with a High Incidence of Gallbladder Cancer.

Our previous study detected aflatoxins in red chili peppers from Chile, Bolivia, and Peru, each of which have a high incidence of gallbladder cancer (GBC). Since the aflatoxin B1 concentration was not so high in these peppers, it is important to clarify the presence of other mycotoxins. Here we attempted to determine any associations between the concentrations of aflatoxins and ochratoxin A (OTA) in red chili peppers, and the corresponding GBC incidences. We collected red chili peppers from three areas in Peru: Trujillo (a high GBC incidence area), Cusco (an intermediate GBC incidence area), and Lima (a low GBC incidence rate), and from Chile and Bolivia. Aflatoxins and OTA were extracted with organic solvents. The concentrations of aflatoxins B1, B2, G1, and G2, and OTA were measured by high-performance liquid chromatography. The values obtained were compared with the incidence of GBC in each area or country. All of the red chili peppers from the three areas showed contamination with aflatoxins below the Commission of the European Communities (EC) recommended limits (5 µg/kg), but the OTA contamination of two samples was above the EC recommended limit (15 µg/kg). The mean concentrations of OTA in the peppers from Chile (mean 355 µg/kg, range <5-1,059 µg/kg) and Bolivia (mean 207 µg/kg, range 0.8-628 µg/kg), which has a high incidence of GBC, were higher than that in Peru (14 µg/kg, range <5-47 µg/kg), which has an intermediate GBC incidence. The OTA contamination in the red chili peppers from Chile, Bolivia, and Peru was stronger than that of aflatoxins. Our data suggest that OTA in red chili peppers may be associated with the development of GBC.

Effects of selenium on gallbladder carcinogenesis induced by an intracholecystic 3-methylcholanthrene beeswax pellet in female Syrian golden hamsters.

This study represents the first report of the effects of selenium (Se) on chemically induced gallbladder carcinogenesis in hamsters. A total of 100 female Syrian golden hamsters was randomly assigned to four groups, which groups of 25 hamsters were given ad libitum drinking water containing either 0.0, 0.5, 2.0 or 4.0 ppm Se (as sodium selenite) for 24 weeks. Initiation was performed at week 4 by the insertion of a Beeswax pellet containing 3-methylcholanthrene (3-MC) into the gallbladder. The incidence of total malignant tumors at the end of the study (24 weeks) was 88, 75, 81 and 82% in the 0.0, 0.5, 2.0 and 4.0 ppm Se groups, respectively. All the cases of carcinoma but two were considered to develop through the sequence from dysplasia to carcinoma in situ (CIS) and from CIS to adenocarcinoma of invasive type. The incidence of CIS was significantly lower in hamsters treated without Se than in those treated with Se (P < 0.05). On the other hand, the incidence of invasive adenocarcinoma was significantly higher in the former than in the latter (P < 0.05). These results were summarized that Se might retard the progression of hamster gallbladder carcinogenesis induced by a 3-MC beeswax pellet.

Effect of secretin on pancreatic carcinogenesis in the hamster model.

Secretin (SEC) given s.c. to hamsters at a dose of 100 clinical units/kg body weight by 6 injections each 30 min apart, inhibited induction of pancreatic cancer when it was given prior to or simultaneously with a single dose of N-nitrosobis(2-oxopropyl)amine (BOP), but was ineffective in modifying tumor incidence when it was administered after BOP. The incidence of gall bladder and common bile tumors were not influenced, regardless of the time of SEC injections, implying that the inhibitory effect of this secretagogue is pancreas specific. However, a treatment schedule in which BOP and SEC were given weekly for 20 weeks did not alter the incidence of pancreatic tumors.

Combined oral contraceptives and gallbladder cancer. The WHO Collaborative Study of Neoplasia and Steroid Contraceptives.

The relationship between combined oral contraceptives and primary gallbladder cancer was examined in 58 cases and 355 controls participating in an international hospital-based, case-control study. Use of combined oral contraceptives at any time was not associated with the risk of developing gallbladder cancer (RR = 0.6, 95% Cl 0.3, 1.3), and no clear patterns of risks were observed according to the duration of use, or time since first or most recent use. There was also no evidence that oral contraceptives effected risk of gallbladder cancer in women with or without a prior history of gallbladder disease. A history of gallbladder disease or gallstones was a strong risk factor for gallbladder cancer (RR = 2.3,95% Cl 1.2,4.4).

Comparative studies of neoplastic response to a single dose of nitroso compounds. The effect of N-nitrosobis (2-hydroxypropyl)amine in Syrian golden hamsters.

Single s.c. applications of N-nitrosobis(2-hydroxypropyl)amine (BHP) caused carcinogenic effects in adult Syrian golden hamsters at doses as low as 1/40 of the LD50. The tumor spectrum decreased at lower doses, which affected the pancreas more strongly than the liver, whereas higher doses exhibited a more pronounced hepatocarcinogenicity and also induced tumors in the respiratory tract, gallbladder, kidney, and vagina. These results demonstrated a potent pancreatic carcinogenic effect of BHP at the lowest dose level. Hence, the presence of BHP in the environment, even in small quantities, should be regarded as a potential hazard to human health.

Carcinogenic effects in the Syrian golden hamster of N-methyl-N-formylhydrazine of the false morel mushroom Gyromitra esculenta.

N-Methyl-N-formylhydrazine (MFH) was administered in drinking water as a 0.0078% solution to randomly bred Syrian golden hamsters for life beginning at 6 weeks of age. The treatment gave rise to benign and malignant liver cell tumors, malignant histiocytomas and tumors of the gall bladder and bile ducts. The tumor incidence in these four treated tissues was 43, 34, 11,8%, while in untreated controls it was 0, 0, 0, 0%, respectively. Histopathologically, tumors were classified as benign hepatomas, liver cell carcinomas, malignant histiocytomas, adenomas and adenocarcinomas of the gall bladder, cholangiomas, and cholangiocarcinomas.

Carcinogenic effects of 1,2-dibromoethane (ethylene dibromide; EDB) in Japanese medaka (Oryzias latipes).

The carcinogenicity of 1,2-dibromoethane (ethylene dibromide; EDB) was investigated in the Japanese medaka (Oryzias latipes), a small fish species. EDB was administered in water continuously for 97 days to a low concentration group, for 73 days to an intermediate concentration group, and intermittently for 24 h once each week over 97 days to a high concentration group. Medaka were 7 days old at the beginning of the tests. Mean measured EDB concentrations in the ambient water were 0.13 mg l-1, 6.20 mg l-1, and 18.58 mg l-1 in the low, intermediate, and high concentration groups, respectively. Two control groups, one inside and one outside the exposure apparatus, were used. Samples were examined histologically at 24, 36, and 58 weeks from the beginning of the tests. EDB was clearly carcinogenic to medaka in the intermediate and high concentration groups causing (1) hepatocellular adenomas and carcinomas, (2) cholangiomas, (3) chloangiocarcinomas, and (4) gall bladder papillary adenomas and adenocarcinomas. In separate studies, medaka exposed to 1.0 mg l-1 EDB for 2 to 5 weeks had elevated hepatic glutathione S-transferase activities, possibly indicating induction of a pathway that forms the reactive metabolite of EDB in mammals. SDS-PAGE of hepatic cytosolic fractions of EDB-exposed medaka showed a pronounced increase in a band at 26,000 Da, the expected position for GSH-S-transferase. Although little is known about EDB's mechanisms of action, medaka appear exceptionally sensitive to the carcinogenic effects of EDB and could serve as a model test species for studying similar compounds.