Subchronic toxic effects of Fusarium moniliforme and fumonisin B1 in rats and mice.

Fumonisins are mycotoxins produced by the fungi Fusarium moniliforme, F. proliferatum, and other Fusarium species. Fumonisin B1, the most commonly found fumonisin, causes the fatal diseases equine leukoencephalomalacia and porcine pulmonary edema. Fumonisins are suspected human carcinogens because of the extraordinarily high incidences of esophageal cancer coincidentally found in areas of southern Africa and China where F. moniliforme-contaminated corn is consumed as a dietary staple. The subchronic (up to 90 days) effects of F. moniliforme-contaminated corn, corn cultures of this fungus, and purified fumonisin B1 (FB1) in rats and mice were systematically studied to determine target organs, characterize organ-specified lesions, and obtain dose-response data. The liver is a target organ in both species. Serum chemical findings indicative of hepatocellular injury and morphological findings, including apoptosis, appeared qualitatively similar in both species. The kidney is also a target organ in rats, but not mice. Lesions which include apoptosis and cellular degeneration are predominately found in the outer medella. Results of several investigations showed that the kidney was consistently affected at lower doses than the liver. The "no-observed-effect" level for nephropathy in rats was also consistently lower in males than females, suggesting a sex-related difference in nephrotoxic response to fumonisins. Other findings suggest that toxigenesis may be mediated by disruption of de novo sphingolipid biosynthesis. Hepatic and renal sphingolipid profiles, specifically sphinganine concentration and sphinganine-to-sphingosine ratio, were altered in rats fed FB1 at levels that did not cause serum chemical, organ weight, or histopathological evidence of toxicity.

Studies on the reproductive effects of Fusarium moniliforme culture material in rats and the biodistribution of [14C] fumonisin B1 in pregnant rats.

Fumonisins are mycotoxins produced by Fusarium moniliforme and other Fusarium species. They are commonly found in corn and corn-based foodstuffs. Fumonisins inhibit sphingolipid (SL) biosynthesis, alter cellular SL profiles, and thus may affect cell proliferation and differentiation, both of which are important processes for reproduction. However, there are few reports of the effects of F. moniliforme or fumonisins on mammalian reproduction or development. To study the reproductive effects of this fungus, diets formulated with culture material of toxic F. moniliforme strain MRC 826 (CM) to provide 0, 1, 10, or 55 ppm fumonisin B1 (FB1) were fed to male and female rats beginning 9 and 2 weeks before mating, respectively, and continuing throughout mating, gestational, and lactational phases of the study. CM caused nephropathy, typical of FB1, in males fed > or = 10 ppm and females fed 55 ppm FB1. No significant reproductive effects were found in males (n = 12/group), dams, and fetuses examined on gestation day 15 (G15) (n > or = 8/group), or dams and litters through day 21 postpartum (n > or = 9/group). Litter weight gain in the 10 or 55 ppm FB1 groups was slightly decreased; however, gross litter weight and physical development of offspring were not affected. Altered SL ratios indicative of fumonisin exposure, specifically increased sphinganine to sphingosine ratios, were found in the livers of dams from the 55-ppm FB1 group on G15. However, SL ratios of abdominal slices, containing liver and kidney, of fetuses from control and high-dose litters did not differ. In a second experiment, two dams were injected intravenously on G15 with 101 micrograms [14C]FB1 (3.179 x 10(5) dpm). After 1 hr, which allowed for ca. 98% of the dose to be cleared from the maternal blood, negligible amounts of radioactivity were found in the fetuses. Together, these results indicate that the CM, and by inference FB1, did not have significant reproductive effects at doses which are minimally toxic, and further suggest that little in utero FB1 exposure occurred through G15.

Subchronic toxicity of fumonisin B1 to male and female rats.

Fumonisins are a class of mycotoxins produced by Fusarium moniliforme and other Fusarium spp. These compounds are widely distributed in corn. Equine leukoencephalomalacia, pulmonary oedema in swine, and nephrotoxicity, hepatotoxicity and liver cancer in male rats, all of which are caused by toxic F. moniliforme, have been experimentally reproduced using fumisin B1 (FB1) (ca 90-94% purity). To investigate the effect of purified (> or = 99% purity) FB1, to compare the effects of FB1 in males and females, and to obtain dose-response information for FB1, three rats per sex were fed diets containing 0, 15, 50 or 150 FB1 for 4 weeks. Serum chemical, organ weight and histopathological evidence showed that 150 mg/kg FB1 was hepatotoxic in both sexes. Nephrosis was found in males fed > or = 15 mg/kg and females fed > or = 50 mg/kg FB1. Altered sphingolipid profiles, specifically increased free sphinganine concentrations and increased sphinganine:sphinogosine ratios, were found in the liver, kidney, serum and urine of FB1-fed rats. These findings support the hypothesis that in vivo toxicity caused by fumonisins may result from altered sphingolipid metabolism.

Subchronic feeding study of the mycotoxin fumonisin B1 in B6C3F1 mice and Fischer 344 rats.

Fumonisin B1 (FB1) is a mycotoxin produced by Fusarium moniliforme, a common fungus which occurs naturally on corn, and other Fusarium species. FB1 and other fumonisins are now recognized as having potentially important animal and human health implications. However, few toxicological data are currently available. Male and female B6C3F1 mice and Fischer 344 rats were fed diets containing 0, 1, 3, 9, 27, or 81 ppm FB1 (> or = 98% purity) for 13 weeks. No differences in behavior or appearance, body weight or food consumption between control and FB1-fed groups were found. In mice, hepatopathy and altered serum chemical profiles indicative of hepatotoxicity were found in females fed the 81 ppm diet. No adverse effects were found in female mice fed < or = 27 ppm FB1 or in male mice at any dietary level studied. In rats, nephrosis involving the outer medulla was found in males fed > or = 9 ppm and, to a lesser degree, in females fed 81 ppm FB1, while decreased kidney weight was found in both sexes at dietary levels > or = 9 ppm FB1. Although the liver is a target organ of FB1 in rats, hepatotoxicity was not found in rats fed diets containing up to 81 ppm FB1 for 90 days. Thus, FB1 was toxic to both species following subchronic oral exposure, although significant interspecies differences in the no observed effect levels and organ-specific responses were found.

Dietary fumonisin B1 induces disruption of sphingolipid metabolism in Sprague-Dawley rats: a new mechanism of nephrotoxicity.

Fumonisins are potent inhibitors of sphingolipid biosynthesis produced by several Fusarium species. Consumption of corn or corn products infected with F. moniliforme, or high levels of fumonisins, is associated with several animal diseases. In a 4-wk feeding study, the concentration of fumonisin B1 that caused nephrotoxicity in Sprague-Dawley rats was much less than that required to cause hepatotoxicity. This retrospective study shows a close correlation between the extent and severity of ultrastructural lesions and the degree of disruption of sphingolipid metabolism. The kidney was more sensitive to fumonisin B1-induced disruption of sphingolipid metabolism than liver with significant elevation of free sphingosine, free sphinganine, and the free sphinganine:free sphingosine ratio in rats fed 15, 50 and 150 micrograms/g fumonisin B1. Accumulation of free sphinganine and elevation of the free sphinganine:free sphingosine ratio in urine closely reflected the changes that occurred in kidney. The accumulated sphinganine and elevation of the free sphinganine:free sphingosine ratio was associated with accumulation of cells in urine. Thus, urine rather than serum is the fluid of choice for detecting elevated free sphingoid bases generated as a consequence of fumonisin-induced kidney damage.

Levels of fumonisin B1 in corn naturally contaminated with aflatoxins.

Aflatoxins and fumonisin B1 are hepatotoxic and carcinogenic metabolites produced by Aspergillus flavus and Fusarium moniliforme, respectively. These fungi are common natural contaminants of corn, and both aflatoxins and fumonisin B1 have been implicated as aetiological agents in animal and human diseases. To determine whether these mycotoxins co-exist on corn under natural conditions, 28 samples from the 1991 Georgia (USA) corn crop were assayed for (total) aflatoxin and fumonisin B1. 27 samples were positive for aflatoxin, 24 samples were positive for fumonisin B1, and 23 samples had detectable levels of both. In the positive samples, the mean aflatoxin concentration was 73 ppb (SD = 86), and the average fumonisin B1 concentration was 0.87 ppm (SD = 0.65). A correlation between aflatoxin and fumonisin B1 concentrations was not evident. None the less, these results demonstrate that exposure to both mycotoxins can occur simultaneously by consumption of co-contaminated corn.

Neurotransmitters in rats fed fumonisin B1.

Fumonisin B1, a toxin produced by Fusarium moniliforme, has been associated with a neurotoxic syndrome in horses known as equine leukoencephlomalacia. Previous investigations showed that F. moniliforme cultured on corn and incorporated into rat chow increased brain 5-hydroxyindoleacetic acid (5HIAA) and 5HIAA: serotonin (5HT) ratios in these animals. Therefore, this study was undertaken to determine whether fumonisin B1 would produce related neurochemical effects in the brain and pineal gland of male and female rats. Rats were fed fumonisin B1 at 15, 50, and 150 ppm for 4 weeks. No differences occurred in brain concentrations of norepinephrine, dopamine, 3,4-dihydroxyphenylacetic acid, 3-methoxytyramine, homovanillic acid, 5HT, 5HIAA, and the 5HIAA to 5HT ratios in either male or female rats, nor where there differences between the sexes. When compared across sexes, the norepinephrine to dopamine ratios were decreased (P < 0.05) in the 150-ppm-treated animals. This may suggest a fumonisin B1-induced imbalance in brain norepinephrine and/or dopamine. No differences were observed in pineal norepinephrine, 5HT, 5HIAA, and the 5HIAA to 5HT ratios. Since fumonisin B1 failed to duplicate the effects of the F. moniliforme-induced imbalances in 5HT and 5HIAA metabolism in the brains of rats, other mycotoxins from F. moniliforme may be responsible for these effects.

A preliminary investigation on renal and hepatic toxicity in rats fed purified fumonisin B1.

Fumonisins are metabolites of Fusarium moniliforme and other Fusarium spp. Fumonisin B1 (FB1) was hepatocarcinogenic (50 ppm, > or = 90% purity) when fed to male rats; however, neither the effects of FB1 on females nor the relationship between dietary FB1 levels and toxicity in rats has been reported. Male and female rats (three per sex per group) were fed diets fortified with 0, 15, 50 or 150 ppm FB1 (> or = 99% purity) for 4 weeks. There were no differences in general appearance or behavior among groups and significant differences in weight gain or food consumption were not found. Histopathological examinations and serum chemical profiles, including significant increases in triglycerides, cholesterol, and alkaline phosphatase, confirmed that 150 ppm FB1 was hepatotoxic to both sexes. Cortical nephrosis was found in males fed > or = 15 ppm and females fed > or = 50 ppm FB1. Both hepatic and renal lesions were consistent with those found in rats consuming F. moniliforme-infected corn. Thus, highly purified FB1 is unequivocally capable of inducing the subchronic liver and kidney lesions attributed to F. moniliforme.

Distribution and excretion of [14C]fumonisin B1 in male Sprague-Dawley rats.

[14C]Fumonisin B1 was biosynthetically produced by the addition of [14C]methyl methionine to a liquid culture of Fusarium moniliforme. The labeled toxin was then administered to rats intragastrically in one study and intravenously in another. The rats were killed at intervals up to 96 hr after dosing. In a third study, rats were dosed intragastrically 3 times at 24 hr intervals, and killed at intervals up to 144 hr after the first dose. After intragastric administration, up to 80 percent of the radiolabel was recovered in feces and up to 3% in urine. The remainder of the radioactivity was distributed in tissues, with the liver, kidney, and blood having the highest percentages. The radioactivity appeared to persist in these tissues for the duration of the experiment. This observation was duplicated in rats dosed intravenously, as well as the fact that urinary excretion of systemic [14C]fumonisin B1 takes place. Also observed during the intravenous study was the elimination of up to 35% of the radiolabel in feces, indicating that fumonisin B1 and/or its metabolites undergoes biliary excretion. The results obtained suggest a portion of the fumonisin B1 that is absorbed is persistent in the target organs, liver and kidney, for up to 96 hr.

Alteration of tissue and serum sphinganine to sphingosine ratio: an early biomarker of exposure to fumonisin-containing feeds in pigs.

Fumonisins are a group of naturally occurring compounds produced by the fungus Fusarium moniliforme. They are believed to be the etiologic agent of several animal diseases associated with consumption of corn-based feeds including porcine pulmonary edema. Recently it was shown in vitro that fumonisins are specific inhibitors of sphingosine and sphinganine N-acyltransferases. Inhibition of these enzymes in cultured cells results in the accumulation of free long chain sphingoid bases, specifically sphingosine and sphinganine, and the depletion of complex sphingolipids. In this study, tissues and serum from male SPF pigs fed a nutritionally balanced diet containing corn or corn screenings naturally contaminated with fumonisins for up to 14 days were analyzed for free sphingoid bases and complex sphingolipids. Total fumonisins (B1 and B2) in the diets were analyzed at 0 (< 1), 5, 23, 39, 101, and 175 ppm. Pulmonary edema only occurred at 175 ppm, while histologic liver damage was present at > or = 23 ppm, and serum liver enzymes were significantly elevated at > or = 101 ppm. The results of this study show that free sphinganine is elevated in liver, lung, and kidney, from pigs consuming feeds containing fumonisins at total fumonisin concentrations of 23 ppm or greater. Sphingosine is also elevated in a dose-dependent manner, but to a lesser extent than sphinganine. The consequence of this differential inhibition is that the ratio of sphinganine to sphingosine increases, suggesting that sphinganine N-acyltransferase is the preferred target for fumonisins. Elevation of free sphinganine and free sphingosine in serum paralleled the increases in tissues. Statistically significant increases in the ratio were observed at feed concentrations as low as 5 ppm total fumonisins and in pigs (at higher concentrations) in which other serum biochemistry parameters and tissue morphology were not altered. Elevated ratios were also observed in serum from pigs fed pure fumonisin B1. The sensitivity of the ratio indicates that it could serve as an effective biomarker for consumption of fumonisin-containing feeds. In addition, the data supports the hypothesis that inhibition of sphingosine and sphinganine N-acyltransferase plays an important role in the pathogenesis of animal diseases associated with consumption of feed containing fumonisins.

The application of solid phase extraction to the analysis of tobacco-specific nitrosamines.

A new isolation and separation method has been developed for the alkaloid-related nitrosamines in tobacco, called tobacco-specific nitrosamines (TSNA). The improved isolation procedure involves the extraction by sonication of TSNA from cured tobacco with toluene and an aqueous citrate buffer solution, while purification is achieved by solid phase extraction on small silica cartridges. Interfering extract materials are eluted from the silica cartridges with the toluene and a chloroform-methylene chloride solvent, while subsequent elution with chloroform yields the TSNA in a purified fraction. Gas chromatographic analyses on an SE-54-coated glass capillary column and detection by a nitrogen-phosphorous detector produce very good data on the four TSNAs, which include N-nitrosanatabine (NATB), N-nitrosoanabasine (NAB), 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (called NNK), and N-nitrosonornicotine (NNN). The method is shown to be quantitative, reproducible, and applicable to the determination of TSNA in various tobacco samples, such as stems and lamina of cured tobacco leaves, as well as to smokeless tobacco.

A study on the mutagenicity of tobacco smoke from low-tar cigarettes.

A series of 16 low-tar cigarettes, yielding from 1 to 10 mg of tar, were smoked on a modified cigarette smoking machine that collected both mainstream (MS, inhaled) smoke and sidestream (SS, between puffs) smoke. The SS smoke is the major contributor to environmental tobacco smoke. The collected MS and SS smoke condensates were evaluated for mutagenicity by the Ames test and compared with MS and SS smoke condensates from a high-tar cigarette. Both MS and SS condensates of low-tar cigarettes (LTCs) were tested with the Salmonella strains TA1538 and TA100. Except for three cigarettes, the MS smoke mutagenicities of the LTC smoke condensates were significantly reduced (about 30%) when compared with a control, high-tar (23-mg) cigarette. Opposite results were obtained for the SS smoke condensates, which were more mutagenic (about 20%) than the SS smoke condensate of the high-tar cigarette. Thus, LTC mainstream smoke may be less hazardous to the LTC smoker, whereas LTC sidestream may emit more mutagenic compounds into environmental tobacco smoke, which, through passive inhalation, could affect both smokers and nonsmokers.

Selenium-mediated reduction in the mutagenicity of cigarette smoke.

Cigarette smoke contains carcinogens and mutagens and affects the health of smokers. Recently, increased research has proven the potentially protective activity of selenium (Se) against heavy metal toxicity, cancer, and other health disorders. Accordingly, we have proposed the fortification of tobacco with Se to develop safer cigarettes. As a start in evaluating any biological effects of added Se, we have determined the mutagenicity of inhaled, mainstream (MS) cigarette smoke condensate (CSC), with and without Se, in the preincubation assay of the Ames test. Initially, it was shown that Se, as sodium selenite, was not mutagenic at high concentrations (up to 80 micrograms/plate) with strains TA1538 and TA1978. Subsequently, the effects of different levels of Se, added to MS CSC, were examined with TA98, TA100, and TA1538. On the average, addition of 10 micrograms Se produced mutagenicity reductions of about 50%. Higher levels of added Se yielded further reductions. Cigarette sidestream (SS) smoke, collected between puffs, was also tested. Again, Se added to SS-CSC gave similar reductions, confirming its antimutagenic effect for both mainstream and sidestream smoke.

Identification of polynuclear aromatic hydrocarbons in cigarette smoke and their importance as tumorigens.

Fraction F20, which in other studies was the most tumorigenic neutral fraction of cigarette smoke condensate (CSC), was separated by gel filtration chromatography into refined subfractions for identification of the polynuclear aromatic hydrocarbons (PAH) and for bioassay on mouse skin. Several hundred PAH were positively identified. Subfraction F55. containing most of the carcinogenic PAH as well as numerous unidentified components, was almost as tumorigenic to 7,12-dimethylbenze[a]anthracene (DMBA)-pretreated female outbred CD-1 mice as was F20. When F55 was separated into two parts, the first containing unidentified material (F55A) and the second containing the PAH (F55B), neither was significantly tumorigenic. F55B, combined with two other active fractions from the neutral and the acidic portions of CSC, exhibited a synergistic tumorigenic effect on DMBA-pretreated mice. The results supported the concept that the PAH in cigarette smoke must interact with other components in order to exert a tumorigenic effect.

Mouse skin tumorigenesis and induction of aryl hydrocarbon hydroxylase by tobacco smoke fractions.

Ten fractions separated from the neutral portion of cigarette smoke condensate (CSC) were tested on mouse skin for tumor-initiating activity and for their capacity to induce the enzyme aryl hydrocarbon hydroxylase (AHH). Tumor-initiating activity was confined primarily to the fraction containing more than 90% of the polynuclear aromatic hydrocarbons (PAH) in CSC. One other PAH-containing fraction was active. The combined initiating effect of these fractions was comparable to that of a 40-ppm solution of benzo[a]pyrene (BP), which is about 40 times the BP content of CSC. Some of the neutral fractions that have been demonstrated to cause tumor promotion in mice pretreated with 7,12-dimethylbenz-[aA1ANTHRACENE sere inactive as tumor initiators. The fractions that contained aromatic hydrocarbons induced mouse skin AHH levels twofold to sixfold after a single topical application. AHH-inducing activity was not, however, a reliable indicator of the carcinogenic potential of a fraction.