Robert M. Eppley

Effects of fumonisin B1 on lipid peroxidation in membranes.

Electron spin resonance (ESR)1 spin-label oximetry and spin trapping techniques have been used to study the effect of fumonisin B1 (FB1), an amphipathic mycotoxin on lipid peroxidation in egg yolk phosphatidylcholine (EYPC) bilayers. In the study of the interaction between FB1 and lipid bilayers our results show that fumonisin disturbs the ordering of membranes, enhances oxygen transport in membranes, and also increases membrane permeability. In our model system, lipid peroxidations were initiated by extended incubation of the liposomes, or by inducing Fe2+ ions, UV illumination of H2O2 or ultrasound irradiation. As an indication of the rates of lipid oxidation in EYPC, the consumption of molecular oxygen was studied by monitoring the oxygen concentration in the aqueous phases of the liposomes. Lipid-derived free radicals generated during the oxidation process were measured by a spin trapping method. The incorporation of FB1 in the test samples made the membranes highly susceptible to oxidation. Our results provide the first evidence that the fumonisins appear to increase the rate of oxidation, promote the free radical intermediate production and accelerate the chain reactions associated with lipid peroxidation. The disruption of membrane structure, the enlargement of the relative oxygen diffusion-concentration products, as well as the enhancement effects on membrane permeability, thus provide additional insights into potential mechanisms by which the fumonisins could enhance oxidative stress and cell damage.

Feasibility of Immunodiagnostic Devices for the Detection of Ricin, Amanitin, and T-2 Toxin in Food

Qualitative and quantitative comparisons were conducted of commercially available immunodiagnostic devices for the detection of three select agents with oral LD50 values > or = 0.1 mg/kg of body weight. Ricin (oral LD50 > 1 mg/kg), amanitin (oral LD50 approximately 0.1 mg/kg), and T-2 toxin (oral LD50 > 1 mg/kg) were spiked into beverages, produce, dairy, and baked goods and assayed using commercially available enzyme-linked immunosorbent assays (ELISAs) and lateral flow devices. In all cases, the commercial diagnostic kits successfully detected all three select agents at concentrations below what might be a health concern. The considerable difference between the limit of detection of the immunodiagnostic devices employed (typically < or = 0.020 microg/g) and the amount of the select agent necessary to pose a health threat in a single serving of food facilitated the design of protocols for the high throughput screening of food samples. These protocols entailed simple extraction methods followed by sample dilution. Lateral flow devices and sandwich ELISAs for the detection of ricin had no significant background problems due to the food matrices. Competitive ELISAs, which typically have unacceptably high background reactions with food samples, successfully detected amanitin and T-2 toxin.

Hepatic alterations produced in mice by xanthomegnin and viomellein, metabolites of Penicillium viridicatum

Abstract Xanthomegnin and viomellein obtained from a toxigenic isolate of Penicillium viridicatum were fed to weanling male Swiss mice at dietary concentrations of 448 and 456 mg/kg of feed, respectively. Gross alterations included jaundice, greenish discoloration of the kidney, and small foci of discoloration in the liver. The histologic alterations in the liver were centered about the intrahepatic biliary ducts and included necrotizing cholangitis, periductal edema and pericholangitis, disseminated focal hepatic necrosis, periductal fibrosis, and hypertrophy and hyperplasia of biliary epithelium. The spectrum of hepatic lesions were as previously produced in mice by crude cultural products of P. viridicatum .

Peroxidation of membrane lipids and oxidative DNA damage by fumonisin B1 in isolated rat liver nuclei

Fumonisin B1 (FB1), a contaminant of corn, has been reported to be a hepatocarcinogen in rats. In an attempt to understand its mechanisms of action, a model system of isolated rat liver nuclei was used to determine what effects, if any, FB1 might have on nuclear membrane lipids and DNA. The data suggested that FB1 induced lipid peroxidation concurrently with DNA strand breaks in this in vitro system. Iron and copper had no statistically significant stimulatory effects on these reactions. In addition, the active oxygen scavengers catalase, superoxide dismutase (SOD), mannitol and sodium azide had no significant inhibitory effects on the FB1-induced DNA strand breaks. However, a small but significant reduction in lipid peroxidation by catalase and mannitol was observed. These results suggested that hydroxyl radicals may be the initiators of the nuclear membrane lipid peroxidation, which results in production of peroxyl radicals. In turn, the peroxyl radicals may be responsible for the DNA strand breaks. An alternative explanation is that the hydroxyl radicals, produced close to the DNA-bound metal ions, may induce direct site-specific strand breaks, which are insensitive to the scavengers of active oxygen.

Effects of Fumonisin B1 in Pregnant Rats

Fumonisin B1 (FB1), the major mycotoxin from Fusarium moniliforme, has been implicated as a causative agent in several animal and human diseases. Despite animal toxicity studies and human epidemiological studies of FB1, knowledge of its reproductive effects is scarce. In this study, one of a series of proposed studies that will allow extrapolation to humans, pregnant rats were given oral doses of 0, 1.875, 3.75, 7.5 or 15 mg FB1/kg on gestation days 3 16. Caesarean sections were performed on day 17 or 20, and maternal condition, implantation efficiency, foetal viability and foetal development were measured. Dose-related decreases in overall feed consumption and body weight gain were seen, but only the feed consumption decrease at 15 mg/kg, and the decreased body weight gain at 15 mg/kg on days 0-17 were statistically significant. Foetal body weights at day 17 were similar in control and treated groups; but in day-20 foetuses, female weight and crown-rump length were significantly decreased at 15 mg/kg. FB1 was not teratogenic at the doses tested, and no dose-related effects were seen in either skeletal or soft-tissue development. In day-17 animals, maternal and foetal brain, liver and kidney tissues, and maternal serum were preserved to study the levels of sphinganine (Sa), sphingosine (So), and the Sa/So ratios. Dose-related increases were seen in Sa/So ratios in maternal livers, kidneys and serum. Sa/So ratios of maternal brains were not affected, nor were those of foetal kidneys, livers or brains.

Effects of fumonisin B1 in pregnant rats. Part 2

The developmental toxicity of purified fumonisin B1 (FB1), a mycotoxin from the common corn fungus Fusarium moniliforme, was examined in Charles River rats. Pregnant rats were dosed orally on gestation days 3-16 at 0, 6.25, 12.5, 25 or 50 mg FB1/kg body weight/day. FB1 was not teratogenic at the doses tested. At 50 mg/kg, maternal toxicity (inappetence, emaciation, lethargy, death, resorption of entire litters) and foetal toxicity (increased number of late deaths, decreased foetal body weight, decreased crown rump length, increased incidence of hydrocephalus, increased incidence of skeletal anomalies) were seen. The foetal toxicity observed at 50 mg/kg may be related to maternal toxicity. Histopathological evaluation of tissues from dams of control and all treated groups revealed dose-related toxic changes in kidney and liver tissues. Acute toxic tubular nephrosis was seen in kidneys from all treated groups. Hepatocellular cytoplasmic alteration and individual cellular necrosis of the liver was seen in the two high-dose groups. Sphinganine (Sa) and sphingosine (So) were measured in day-17 adult and foetal tissues. Dose related increases in Sa/So ratios were seen in maternal liver, kidney, serum and brain, but there was no effect on foetal liver, kidney and brain. These data suggest that FB1 does not cross the placenta and further suggest that the observed foetal toxicity is a secondary response to maternal toxicity.

Lack of initiation and promotion potential of deoxynivalenol for skin tumorigenesis in sencar mice

The mycotoxin deoxynivalenol (DON; vomitoxin) was tested for its potential to initiate or promote skin tumours through a two-stage treatment regimen in female Sencar mice. DONs capability for initiation was tested by applying a single topical dose (200 micrograms) followed by multiple treatments of the promoter phorbol 12-myristate 13-acetate (PMA). The test for promotion involved initiation with the carcinogen 7,12-dimethylbenz[a]anthracene (DMBA) followed by multiple DON treatments (50 micrograms). Appropriate control groups were included in the study design. Mice were observed for 26 wk and skin tumours were counted. Results of the study showed that DON was not an initiator or a promoter. When DON was tested as an initiator, there were no statistically significant differences in the number of cumulative tumours or the number of tumour-bearing mice between the DON-initiated/PMA-promoted group and its control, the vehicle-initiated/PMA-promoted group. When DON was administered as a promoter, no tumours were observed. Histopathology of the skin revealed that DON induced a mild diffuse squamous hyperplasia, but there was no progression of the lesion to neoplasia.

Electrospray Mass Spectrometry for Fumonisin Detection and Method Validation

Fumonisins are a structurally related group of mycotoxins, characterized by a 19-20 carbon aminopolyhydroxy-alkyl chain which is diesterified with propane-1,2,3-tricarboxylic acid (tricarballylic acid). These mycotoxins are commonly found in corn and corn-based food products and have been linked to a variety of animal toxicities. The widespread prevalence of fumonisins and the toxicity associated with ingestion has resulted in a number of analytical methods for determining the amount of fumonisins present in foods. Among the most common of these methods are liquid chromatographic (LC) separation with fluorescence detection, enzyme-linked immunosorbent assay (ELISA) and LC/mass spectrometry. LC and ELISA give quantitative results while LC/MS provide quantitative analysis as well as confirmation of identity of the fumonisins.

Lack of transforming activity of fumonisin B1 in BALB/3T3 A31-1-1 mouse embryo cells

The capacity of fumonisin B1 (FB1) to induce morphological transformation of cultured mammalian cells was assessed using BALB/3T3 A31-1-1 mouse embryo cells. FB1 with 90% purity was prepared from Fusarium proliferatum grown on whole corn. Cell growth was not inhibited by 48 hr of exposure at concentrations up to 1000 micrograms/ml. Moderate inhibition was induced by 6 days of exposure. In transformation assays with a 48-hr exposure, increases in transformed foci were observed at some concentrations; however, the responses were not reproducible. Prolonged exposure for up to 4 wk at 10, 100 and 500 micrograms/ml failed to induce increases in transformed foci. Analysis of combined results showed that only the increase induced by a 48-hr exposure at 500 micrograms/ml was significant. A trend test indicated the lack of a dose response for concentrations of 10-1000 micrograms/ml. FB1 seems to lack in vitro transforming activity.