Eivind B. Lillehoj

Synergistic toxic effects of citrinin, ochratoxin A and penicillic acid in mice.

Abstract G. A. Sansing , E. B. Lillehoj , R. W. Detroy and M. A. Miller . Synergistic toxic effects of citrinin, ochratoxin A and penicillic acid in mice. Toxicon14, 213–220, 1976.—The ld 50s in mice of citrinin, ochratoxin A, and penicillic acid injected intraperitoneally were 89, 22, and 100 mg per kg of body weight, respectively. Paired combinations of the mycotoxins, citrinin: ochratoxin A (CI:OA), ochratoxin A:penicillic acid (OA:PA), and penicillic acid:citrinin (PA:CI) elicited synergistic lethal responses. After administration of citrinin, 14C-orotic acid incorporation into both liver and kidney increased significantly by 27 hr with a return to control levels at 51 hr. Treatment with penicillic acid also increased orotic acid incorporation into liver ribonucleic acid (RNA) at 27 hr and at 4 hr in the kidney. Ochratoxin A inhibited orotic acid incorporation into both liver and kidney RNA 6 hr after toxin injection with a subsequent return to control levels at 27 hr. Orotic acid incorporation was inhibited in both kidney and liver 6 hr after treatment with the toxin combination CI:OA, PA:CI stimulated precursor incorporation into kidney RNA at 27 hr and inhibited the function in liver. The OA:PA combination inhibited orotic acid incorporation in both organs 15–27 hr after toxin treatment.

Ochratoxin A-induced porcine nephropathy: Enzyme and ultrastructure changes after short-term exposure

Four pigs were treated with ochratoxin A (800 micrograms/kg) for five consecutive days. Subsequently, urine and bile were collected and kidneys were perfusion fixed unilaterally. Liver and kidney samples were examined for the distribution of ochratoxin A and metabolites in subcellular fractions and the effects of the toxin on protein synthesis and enzyme activities. Ochratoxin A and the hydrolytic product, ochratoxin alpha, were found in urine. Elevated levels of toxin accumulation in kidney (283 ng/g) compared with liver (189 ng/g) and toxin-mediated reductions in protein synthesis and enzyme activities in kidney identified it as a target organ of ochratoxin toxicity. Ultrastructural investigations of kidney in toxin-exposed animals identified a process of condensation of cellular material with disappearance of membranes and continuous desquamation in the lower part of the proximal convoluted tubules. In target cells peroxisomes appeared to have lost membrane integrity and the organelles were leaking materials into the cytosol. Reduction of structural integrity was associated with an increase in the presence of catalase and cyanide insensitive fatty acid oxidase activity in the soluble kidney fractions.

Aflatoxin Occurrence in 1973 Corn at Harvest. II. Mycological Studies

SUMMARY Since aflatoxin is formed in corn in the field before harvest, our objectives were to determine at harvest (a) the amount of Aspergillus flavusinfected corn kernels, (b) the amount of A. flavus spores on the surface of corn, (c) the total amount of fungus-infected kernels, (d) the occurrence of A flavus spores in and on insects from corn reported in the first paper of this series, and (e) the correlation between A. flavus infection and occurrence of aflatoxin. The corn was collected at harvest from seven counties in northeastern South Carolina and dried to less than 13% moisture as quickly as possible. Of the 152 aflatoxin-positive samples, 120 showed one or more kernels internally infected with A. flavus and of the 145 aflatoxin-negative samples, 59 showed infection. Of the 297 samples examined, 276 had one or more kernels with surface contamination of A. flavus spores, and in 75 of the samples every kernel was contaminated. When kernels were surface disinfected, 224 samples (50 kernels each) showed 100% internal mold contamination. One or more kernels of 185 samples were infected with A. flavus; this number represents 60% of the total samples. Of the 375 insects collected and examined for A. flavus from the corn samples, 247 showed A. flavus present. Of the 85 rice weevils, 78 were carrying A. flavus spores and of the other 290 insects, 165 were contaminated. Besides A. flavus, the predominant infecting fungi internally were two species of Penicillium and Fusarium. Members of the Mucorales were rarely seen. The occurrence of Aspergillus flavus Link ex Fries and A. parasiticus Speare growing on corn plants in the field before harvest has rarely been recorded (Taubenhaus, 1920). From 0.02 to 0.09% infection of corn kernels before harvest was reported by Tuite (1961) from Indiana. The highest single instance had a 22% infection by A. flavus. In 1970, Tuite and Caldwell (1971) found an average incidence of 0.4% kernel infection before harvest in corn also in Indiana. The A. flavus incidence in southern Indiana counties was 1.2% as compared to 0.2% in northern counties. Rambo et al. (1974) reported a 0.03 to 0.08% incidence of

Aflatoxin M1 removal from aqueous solutions by Flavobacterium aurantiacum

In liquid cultures growing and stationary phase cells ofFlavobacterium aurantiacum NRRL B-184 eliminated aflatoxin M1. Toxin concentrations of 15µg/ml and 37.5µg/ml interfered with bacterial growth, and at the higher level 4.4µg M1 was removed from the growth medium by a milligram (dry weight) of bacteria. Toxin was completely removed from the liquid medium by incubating 5 × 1010 resting cells per milliliter with 8µg/ml of aflatoxin M1 for 4 h. Attempted recovery of M1 from cells following incubation of the bacteria with the toxin demonstrated that the M1 was essentially nonextractable. Bacterial cells also removed aflatoxin M1 from toxin-contaminated milk.

Enzymes in atlatoxin B1 biosynthesis: Strategies for identifying pertinent genes

Recent work on the aflatoxin biosynthetic pathway is reviewed, with special emphasis on the enzymes of the late stages of the pathway involving conversion of sterigmatocystin (ST) to aflatoxin B1 (AFB1) through an O-methylsterigmatocystin intermediate. Two enzyme activities were discovered in subcellular fractions of cell-free extracts of a mutant strain ofAspergillus parasiticus (SRRC 163): 1)A post-microsomal methyltransferase (MT) catalyzed conversion of ST to OMST, and 2) a microsomal-associated activity (oxido-reductase) converted OMST to AFB1. The 168 KDa, anionic MT was purified to homogeneity and characterized (two subunits, 110 KDa and 58 KDa). Preliminary evidence indicated the presence of a cationic isozyme of the MT in mycelial extracts. The oxido-reductase has been partially purified and characterized. Polyclonal antibodies were prepared to the anionic MT and the enzymes amino acid composition determined. A cDNA library has been constructed from mRNA isolated fromAspergillus parasiticus mycelia during the onset of AFB1 biosynthesis for the purpose of identifying the genes responsible for aflatoxin biosynthesis.

Aflatoxin contamination of maize kernels before harvest

Two maize (Zea mays L.) hybrids with varying degrees of resistance to damage by corn earworm (CEW) (Heliothis zea Boddie) were grown in Iowa, Georgia, and Missouri. Treatments included: introduction of Aspergillus flavus Link ex. Fr. spores onto newly-emerged silks, application of a fungicide as an aqueous spray onto test ears during the first three weeks after flowering, infestation of ears with CEW eggs, and combinations of these variables. CEW larvae were collected from developing ears and examined for the presence of internal A. flavus group propagules. Aflatoxin levels were determined in mature kernels. Toxin concentrations exhibited a distinct regional variation with relatively high levels in Georgia samples, intermediate concentrations in Missouri kernels and low levels in Iowa samples. No treatment effects were noted in Georgia samples but introduction of A. flavus and CEW increased toxin accumulation in Missouri kernels. Although the CEW-susceptible hybrid exhibited a trend towards increased damage by the insect, no treatment-related differences were observed in the presence of the fungus in larvae or in aflatoxin contamination. Fungicide applications did not significantly reduce aflatoxin levels in mature kernels.

Tissue distribution of radioactivity from ochratoxin A-14C in rats

Examination of the distribution of radioactivity in rat tissues during the first 24 hr after administration of ochratoxin A-14C demonstrated maximum accumulation in stomach and kidney. The highest counts were observed in stomach, lung, kidney, thymus, spleen and heart during the first 6 hr after treatment, whereas the brain, liver muscle, duodenum, jejenum, ileum, and cecum exhibited the greatest counts at 18 hr after toxin exposure.

Lethality in mice of double-stranded ribonucleic acid from virus-like particles of Penicillium stoloniferum

Abstract The ld 50 in mice of intact virus-like particles (VLP) from Penicillium stoloniferum NRRL 5267 was greater than 240 mg/kg body weight, and their component double-stranded ribonucleic acid (dsRNA) was 34.0 mg/kg. Judged by decreases in ld 50 values, administration of sublethal amounts of actinomycin D or cycloheximide simultaneously with sublethal amounts of intact VLP or mycoviral dsRNA enhanced lethality in mice. A 2400-fold increase in the letahlity of intact VLP occurred in mice treated with actinomycin D and a 65-fold increase with cycloheximide and VLP. Increases in lethality of 850-fold with actinomycin D and 10-fold with cycloheximide were determined when mice were treated simultaneously with either antibiotic and mycoviral dsRNA. Similar lethality effects were observed in mice simultaneously treated with polyriboinosinic-polyribocytidylic acid and either of the antibiotics. Animals treated at zero-time with mycoviral dsRNA and subsequently treated at 1, 2, 4, 8, and 12 hr with actinomycin D generally exhibited a reduced toxic response after the 4-hr injection with actinomycin D. Animals treated at zero-time with actinomycin D and subsequently with mycoviral dsRNA also exhibited a reduced toxic response after the 4-hr injection with the nucleic acid.

Immunospecific binding of aflatoxins to human immunoglobulins in vitro

The aflatoxins are a family of complex coumarins produced by species of Aspergillus that are toxic and carcinogenic in animals. Four of the naturally occurring aflatoxins (B1, B2, G and G2) were incubated in vitro with whole human serum and subsequently tested for effects on antigenicities and electrophoretic mobilities of the proteins. Immunoelectrophoresis showed deviations of some of the precipitin arcs from normal patterns. Double diffusion experiments with affinity-isolated antibodies that are specific for the heavy chains of immunoglobulins IgA, IgG and IgM showed that the antigen-antibody binding sites of the heavy chains of IgA and IgM were blocked after incubation, but not those of IgG. These results are of interest regarding the specificity of immunochemical reactions in vitro that might be linked to specific immunological responses in diseases mediated by these toxins.

Carbon-13 nuclear magnetic resonance assignments and biosynthesis of ochratoxin A

Abstract The carbon-13 chemical shifts have been assigned to all the carbons in the isocoumarin portion of ochartoxin A. Incorporation of carbon-13 enriched acetate was used to confirm the biosynthesis of ochratoxin A.