Mutagenesis, Vol. 15, No. 6, 469-471,
November 2000
© 2000 UK Environmental Mutagen Society/Oxford University Press
Assessment of in vitro mutagenicity in Salmonella and in vivo genotoxicity in mice of the mycotoxin fumonisin B1
Laboratory of Genetics, Faculty of Chemistry and Biology, University of Santiago de Chile, Casilla 40, Correo 33, Santiago and 1 Laboratory of Genetic Toxicology, Faculty of Chemical and Pharmaceutical Sciences, University of Chile, Santiago, Chile
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Abstract |
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Fumonisin B1 (FB1), a mycotoxin produced by Fusarium moniliforme, is a contaminant of cereals with various and complex cellular effects. FB1 induces liver cancer in rats and has been linked to esophageal cancer in South Africa and China. The mechanisms of FB1-induced carcinogenesis are uncertain and the information on FB1 mutagenic properties is limited and controversial. FB1 contamination levels in maize and wheat from Chile were found to be similar to those in other countries. FB1 was devoid of activity in gene mutation assays with Salmonella typhimurium strains TA100, TA102 and TA98. However, i.p. injection of FB1 induced an increased frequency of micronuclei in mouse bone marrow polychromatic erythrocytes at 25 and 100 mg/kg. We conclude that FB1 induces in vivo genotoxicity in the absence of in vitro mutagenicity in Salmonella.
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Introduction |
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Fumonisin B1 (FB1), the major metabolite produced by Fusarium moniliforme, is a mycotoxin found in important amounts in cereals from different countries, principally in maize and wheat. Fumonisin's cellular effects are complex and may differ between species and organs. FB1 is structurally similar to sphingosine, a precursor of sphingolipids. It can act as a ceramide synthase inhibitor (Wang,E. et al., 1991
; Schroeder et al., 1994) producing: a block of sphingolipid biosynthesis, disruption of cellular lipids, fatty acid accumulation and cell proliferation. FB1 and lipid bilayers interact, disrupting the structure of membranes, enhancing oxygen transport in membranes and also increasing membrane permeability (Yin et al., 1998).
FB1 causes liver cancer in rats, acting as a cancer promoter in a short term assay (Gelderblom et al., 1988) and as a cancer initiator at high levels of chronic exposure (Gelderblom et al., 1991, 1994). The mechanisms of FB1-induced carcinogenesis are uncertain and the information on FB1 mutagenic properties is limited and controversial. For some authors fumonisins are the only carcinogenic mycotoxins that do not directly produce DNA damage (Wang and Groopman, 1999) while others reported that FB1 can induce chromosomal aberrations in primary cultured rat hepatocytes (Knasmuller et al., 1997). In cell culture FB1 induces oxidative DNA damage, lipid peroxidation, cell cycle arrest and altered levels of methylated bases (Abado-Becognee et al., 1998; Mobio et al., 2000). To assess the role of genotoxicity in FB1-induced carcinogenicity we assessed the in vitro mutagenicity of FB1 using three Salmonella strains that included TA102, which is susceptible to oxidative DNA damage. In vivo gentoxicity was assessed using the mouse bone marrow micronucleus assay.
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Materials and methods |
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Chemicals, media and bacterial strains
FB1, methyl methanesulfonate (MMS), dimethylsulfoxide (DMSO) and benzo[a]pyrene (B[a]P) were purchased from Sigma Chemical Co. (St Louis, MO). Sodium azide (SA), S9 rat liver fraction and bacterial strains were from Moltox (Boone, NC).
Extraction of FB1 from and determination in maize
The samples were obtained from different parts of Chile. They included wheat, frozen maize (purchased from local markets) and dried maize. Fumonisin extraction was performed using the commercial FumoniTest from Vicam (Watertown, MA). Briefly, the mycotoxin was extracted with methanol:water (80% v/v) in a Waring blender and the extract filtered and washed with buffer solution (0.43 M NaCl, 0.06 M NaHCO3 and 0.1 ml Tween 20) and then filtered through a microfiber paper. The crude extract was passed through a fumonisin monoclonal antibody affinity column. The fumonisin was eluted with pure methanol and measured in a calibrated fluorometer (Tor Bex FX-100). The limit of detection of this method was 0.25 mg/kg.
Salmonella typhimurium Ames assay
The test was carried out according to the protocol of Maron and Ames (1983) and Pérez-Alzola and Santos (1997). Various doses of FB1 were added to 2 ml of top agar, with or without S9 mix, and 200 µl of three different S.typhimurium strains (TA100, TA102 or TA98). After incubation of the plates for 48 h the number of His+ revertant colonies was counted. All experiments were done in duplicate. SA, MMS and B[a]P were used as positive controls. Student's two tailed t-test was used to test for statistical significance.
Micronucleus test
The mouse bone marrow micronucleus test was carried out as previously reported (Ellahueñe et al., 1994). Groups of six CF1 male mice received an i.p. dose of 25 or 100 mg/kg FB1 and after 30 h the animals were killed and bone marow samples were prepared for determination of micronucleus frequency. For each animal, 2000 polychromatic erythrocytes (PCE) were scored and cytotoxicity was measured as the ratio PCE/NCE (normochromatic erythrocytes). MMS and DMSO (0.05%) were used as positive and negative controls, respectively. One-way ANOVA was used to calculate levels of statistical significance.
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Results |
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Levels of FB1 in Chilean cereal samples
All samples of maize contained detectable levels of FB1, while some wheat samples were free of FB1 or had levels below the limit of detection (Table I
). The levels of FB1 were typical for these kinds of cereals in South and Central America (2–5.4 mg/kg).
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Mutagenicity in Salmonella and genotoxicity in mouse bone marrow
FB1 was assessed for in vitro mutagenicity using three Salmonella tester strains. The results of the mutagenicity assay with the Ames Salmonella test are summarized in Table II
. All FB1 doses tested were devoid of activity in the Salmonella/microsome assay in the three strains used.
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To assess the in vivo genotoxicity of FB1 we used the bone marrow micronucleus assay. MMS was the positive control. The results obtained with the two FB1 doses tested in the mouse bone marrow micronucleus assay are summarized in Table III
. The low dose of FB1 (25 mg/kg) was more effective at inducing bone marrow micronuclei (P < 0.001) than the 100 mg/kg dose (P < 0.05) compared with the negative control.
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Discussion |
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In Chile, as in many other Latin American countries, maize and wheat are important food sources. Our diet consists in a great part of fresh maize and maize-based food products, as well as of large amounts of bread prepared with wheat flour. FB1 contamination in wheat and maize from Chile showed levels (Table I
) similar to those reported previously for other countries. In the UK, maize-based retail products presented a range of 0.011–2.124 mg/kg in FB1 contamination. In the USA, maize-based food products were found to contain between 0.2 and 7.45 mg/kg (Castello et al., 1998). In a study of 349 maize samples from 18 countries world wide, 92.5% of the samples contained FB1, the average contamination level being 1.359 mg/kg (de Nijs et al., 1998). In line with these data, in Chile all the samples of maize (16, frozen and dried) that we analyzed were positive for FB1 contamination, containing even higher levels than the international average (Table I). Although no convincing evidence of causality has been presented to date, an association has been suggested between FB1 exposure with esophageal cancer rates in South Africa and China (for a review see Turner et al., 1999). Chilean levels of FB1 in maize are higher than those reported for China, where no positive correlation could be found (Yoshizawa, 1994). Increased numbers of samples and evaluation of FB1 levels from different regions in Chile are needed in order to determine whether a correlation exists between FB1 contamination and geographical distribution of this cancer.
Even though the fumonisins were suspected of being carcinogenic mycotoxins several years ago (Gelderblom et al., 1988), a possible carcinogenic mechanism is still uncertain. Information on the mutagenic properties of FB1 is limited and controversial (Turner et al., 1999) and for some authors fumonisins are the only mycotoxins with carcinogenic potency that are not direct DNA-damaging agents (Wang and Groopman, 1999).
According to our results (Table II) and those of Knasmuller et al. (1997), FB1 is not mutagenic in the Salmonella test. This mycotoxin does not induce base pair substitutions (TA100 and TA102) or frameshift mutations in the Ames test. The lack of mutagenicity in TA102 suggests that FB1 does not induce point mutations at either GC or AT pairs and does not induce oxidative DNA damage resulting in mutant cells (Levin et al., 1982). In the bone marrow micronucleus test, the results indicate that the PCE micronucleus frequency was significantly elevated above controls at the lower dose (25 mg/kg) more than at the higher dose (100 mg/kg). Similar results were obtained in primary cultures of rat hepatocytes, where the results with FB1 were inconclusive at higher exposure levels without clear dose–response effects being established (Knasmuller, 1997). The cause of the decrease in PCE micronucleus frequency following i.p. injection of FB1 is uncertain and indicates that careful dose–response studies assessing micronucleus induction and other genotoxic end points are needed to define potential risk to humans.
To date, only limited evidence of FB1-induced genotoxicity is available (induction of chromosomal breaks in primary cultured rat hepatocytes; Knasmuller, 1997), although the underlying mechanism is still unclear. Considering the chemical structure of FB1, similar to that of sphingosine, we suggest that genotoxic activity is likely to be a secondary effect. Some authors have suggested that it might be possible that the chromosome-breaking effect of FB1 is causally related to an increase in lipid peroxidation (Abado-Becognee et al., 1998). Furthermore, FB1 produces activation of mitogen-activated protein kinase, a central enzyme in the signal transduction pathways that modulate cell proliferation (Wattenberg et al., 1996).
The present findings demonstrate a lack of in vitro FB1-induced mutagenicity in Salmonella tester strains. In the bone marrow of mice, FB1 induced weak genotoxicity relative to the positive control MMS. Genotoxicity in the bone marrow was higher at the lower dose tested than at the higher dose, suggesting a saturation of genotoxic effects at the lower exposure level. In summary, the lack of in vitro mutagenicity in Salmonella and the weak genotoxicity observed in mouse bone marrow support an indirect mechanism of genotoxicity for FB1-induced carcinogenicity.
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Acknowledgments |
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We thank Dr Leslie Recio for editorial assistance with this manuscript. This work was supported by FONDECYT (1990212), FONDECYT I.R. (7990077) and DICYT-USACH.
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Notes |
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2 To whom correspondence should be addressed. Tel: +56 2 681 1381; Fax: +56 2 681 2108; Email: maranda{at}lauca.usach.cl
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References |
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Received on February 1, 2000; accepted on June 30, 2000.
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