A.Wallace Hayes

Evaluation of the mutagenic potential of mycotoxins using Salmonella typhimurium and Saccharomyces cerevisiae

The mutagenic effects of fiteen mycotoxins on Salmonella typhimurium strains TA1535, TA1537 and TA1538 and Saccharomyces cerevisiae strain D-3 were tested. Only aflatoxin B1 and sterigmatocystin were mutagenic. Both were active against S. typhimurium strain TA1538 and S. cerevisiae strain D-3; however, both required activation by the hepatic S-9 enzyme preparation. A positive correlation between the other mycotoxins reported to be carcinogenic and the two in vitro test systems employed was not demonstrated in our hands.

Acute toxicity of penitrem A in dogs

Abstract The effect of ip administered penitrem A, a tremorgenic fungal metabolite, on dogs of both sexes and mixed breeding was determined by serum tests, by observation of clinical signs and survival times, and by evaluation of gross and microscopic lesions. Alkaline phosphatase, glutamic-oxaloacetic transaminase, lactic dehydrogenase and creatinine phosphokinase activities increased; survival time varied in relation to dose of mycotoxin administered. Tremors appeared as early as 10 min after dosing and ended in clonic or tetanic convulsions in dogs receiving 0.5 mg/kg or more of penitrem A. Blood uric acid and cholesterol exceeded the normal range. Gross pathologic changes frequently observed were subserosal and submucosal hemorhages in some organs of the thoracic and peritoneal cavities. Dose-related liver damage varied from massive necrosis in dogs receiving 2.5–5.0 mg/kg of penitrem A to no pathologic alterations in dogs given 0.125 mg/kg. Treatment with pentobarbital was successful for penitrem A poisoning if administered within 1–2 hr after exposure to the mycotoxin.

Excretion and tissue distribution of radioactivity from rubratoxin B-14C in mice and rats

Abstract Excretion patterns of 14 C derived from ip doses of radiolabeled rubratoxin B were investigated in mice and rats. Both species excreted 40–50% of the administered radioactivity during the 24 hr following administration. A major excretory route of the radioactivity was respiratory CO 2 , which accounts for nearly 30% of the administered 14 C in mice and for nearly 40% in rats. A further 6–9% was excreted in the urine. Less radioactivity was present in feces. Radioactivity derived from rubratoxin B was present at maximum levels in livers and kidneys 1 hr after administration, with smaller amounts in other organs. The concentration of radioactivity was greater in liver than in other tissues. This finding is associated with the relative tissue specificity of rubratoxin B as a hepatotoxin. In addition, large amounts of radioactivity were found in the carcass and in circulating blood.

Effect of aflatoxin B1, ochratoxin and rubratoxin B on a protozoan,Tetrahymena pyriformis HSM

SummaryRubratoxin B is more inhibitory to the growth ofT. pyriformis than is either aflatoxin B1 or ochratoxin. Only a marginal effect on cell respiration was exerted by these mycotoxins. The presence of the divalent ions, Mg++, Ca++ and Fe++, did not reduce the toxicity of rubratoxin B. A bioassay sensitive to 1 to 5 μg/ml rubratoxin B has been developed employingT. pyriformis.

Effect of rubratoxin and aflatoxin on oxygen consumption of Krebs cycle intermediates

The in vitro and in vivo effects of rubratoxin B and aflatoxin B1 were examined using whole homogenates and mitochondria from livers of various animal species. At a concentration of 3.28 × 10−4 m, rubratoxin inhibited, in mice, the in vitro oxygen uptake of Krebs cycle intermediates, particularly citrate and succinate. Inhibition was greater in males. Inhibition of oxygen uptake in brain and ileum homogenates was greater than in liver homogenates. Succinate was the only Krebs cycle intermediate inhibited by rubratoxin to a greater extent in vitro than in vivo. A varied species susceptibility to poisoning by rubratoxin also was observed. n nAflatoxin inhibited oxygen uptake in vivo but not in vitro in all species examined except weanling rats. Rubratoxin was the inhibitory agent in in vitro mixtures of rubratoxin B and aflatoxin B1. Neither mycotoxin had an in vitro effect on P/O ratios or on glycolysis in liver homogenates from male mice.

Acute toxicity of rubratoxin B in dogs

Abstract The effect of ip administered rubratoxin B on dogs of both sexes and mixed breeding was determined by serum tests, by observation of clinical signs and survival times and by evaluation of gross and microscopic lesions. Glutamic-oxaloacetic transaminase, lactic dehydrogenase and alkaline phosphatase activities and survival time varied in relation to duration of exposure and total dose of mycotoxin administered. Results of the plasma enzyme tests were useful in estimating hepatic cell necrosis. Clinical signs included anorexia followed by dehydration, somnolence, diarrhea and jaundice. Gross pathologic changes frequently observed were subserosal and submucosal hemorrhages in some of the organs of the thoracic and peritoneal cavities, and mottled livers. The severity of the lesions was dependent on the amount of toxin administered. The lesions in animals injected with a nonlethal dose (0.5 mg/kg) of the toxin included bile stasis and early degenerative changes of hepatic cells without renal damage. The changes in the liver and kidneys from a dog injected with a lethal dose (3.0 mg/kg) consisted of extensive hepatic necrosis and mild degenerative changes in renal tubular epithelium. There was no evidence of fatty change within hepatocytes. No lesions were observed in dogs receiving multiple sublethal doses of rubratoxin B.

Effect of mycotoxins on uptake and degradation of [125I] albumin in mouse liver and kidney lysosomes.

Some mycotoxins have been evaluated with respect to effects on the reticuloendothelial function of uptake and degradation of a soluble, denatured protein in phagolysosomes isolated from mouse liver and kidneys. Toxins were dissolved in dimethylsulfoxide and administered intraperitoneally. Particulate fractions isolated from liver and kidney homogenates were assayed for proteolytic activity within osmotically active particles (phagolysosomes). Treatment with citrinin, penitrem A, streigmatocystin, and zearalenone produced inhibition of proteolysis in kidney but not in liver phagolysosomes. Moniliformin and T-2 toxin had no detectable effect on lysosome formation or function in either tissue. The phagolysosomal toxicity of these compounds appeared to be more toward kidney than liver. Both sterigmatocystin and zearalenone caused an increase in kidney phagolysosomal fragility at early times after treatment, and all agents except penitrem A caused a decrease in uptake of labeled albumin into kidney cells. Citrinin labelized kidney phagolysosomes in vitro but not in vivo, while only citrinin and zearalenone inhibited in vitro preparations of cathepsin.

Effect of fungal toxins on uptake and degradation of formaldehyde-treated 125I-albumin in mouse liver phagolysosomes

Abstract Aflatoxin, ochratoxin and rubratoxin are hepatotoxic metabolites produced by fungi. These mycotoxins have been evaluated with respect to effects on the reticuloendothelial function of uptake and degradation of a soluble, denatured protein by phagolysosomes isolated from mouse liver and kidneys. The toxins were dissolved in dimethylsulfoxide and administered intraperitoneally. In addition, rubratoxin B in corn oil was given orally. Particulate fractions isolated from liver and kidney homogenates were incubated at 35° and assayed for proteolytic activity within osmotically active particles (phagolysosomes). Osmotically active particles containing labeled protein were present in liver homogenates 3–4 hr after animals received 2–5 mg kg of rubratoxin, but these particles exhibited little or no proteolytic activity. Smaller doses of rubratoxin B resulted in increased intralysosomal digestive capacity; however, the relative quantity of osmotically active particles was less than in dimethylsulfoxide-treated controls. No effect was noted on phagolysosome formation or function in the kidney except at high doses. Similar results were obtained when 100 mg kg of rubratoxin B was administered orally to mice. Rubratoxin B had no effect on proteolytic activity in a crude mouse liver cathepsin preparation. These observations are interpreted as an interference by rubratoxin B in the fusion of lysosomes with phagosomes. Aflatoxin B1 and ochratoxin had no effect on uptake and degradation of protein in phagolysosomes.

EFFECTS OF PRENATAL ADMINISTRATION OF PENICILLIC ACID AND PENITREM A TO MICE

METABOLITES of certainmolds growingon foodstuffs causetoxicdiseasesinman and animals;however,anunderstanding oftherelationship betweenthemold,thefoodandthe diseasehas onlynow beguntodevelop(NEWBERNE, 1974).Pencillia and Aspergillia are especially importantas contaminatingorganismsbecausecertainspeciescan grow and producetoxicmetabolites on agricultural commoditiesunderwidelydifferent conditions of moistureand temperature .Effectsof mycotoxinson biological systemsarenumerous; rangingfrom suddendeathtotumor formationfollowingingestion ofsmallquantities of toxinoveranextendedperiodoftime.The economicsignificance ofmycotoxicoses, though unknown,certainly ishigh.The presenceofmycotoxinsinfeedstuffs probablycauses,in additiontoacuteeffects, economiclossesby affecting suchimportantaspectsasweight gain,impairmentof resistance and immunitytodisease, and damage totheoffspring (PIER, 1973). A number offungithatgrowon standingcropsorstoredfeedarecapableofproducing toxicmetabolites .Two suchcompounds arepenicillic acidand penitremA .Penicillic acid, 3-methoxy-5-methyl-4-oxo-2,5-hexadienoic acid,first isolated by AISBERG andBLACK (1913), isa secondarymetaboliteproducedby speciesofPenicillium and Aspergillus (OXFORD etal.,1942).Penicillic acidistoxictomammals (HAYES etal.,1977),hasantibiotic activity (OXFORD, 1942;OXFORD etal.,1942),andhasbeenreportedtobecarcinogenic toratsand mice(DICKENS and JONES, 1961;DICKENS and JONES, 1965).Thismycotoxinoccursnaturallyinseveralagricultural products,includingmoldy tobacco,cornand peas(WILSON, 1976). Severalspecies ofPenicillium produceametabolite, penitremA (C87H 44NO,,Cl), capable ofcausingsustainedtremors,convulsionand evendeathinanimals(WILSON and HAYES, 1973). P.crustosunt and P.palitans, two tremorgenicspecies, havebeen implicatedin mycotoxicosesinvolvingcontaminatedfeedstuffs (WILSON etal.,1968;CIEGLER, 1969). Cows, sheep,mice,rats,chickens, rabbits, guineapigs,dogsand hamstersareamong the animalssusceptible toitsneurotoxic properties (CYSEWSKI etal.,1975;WILSON etal.,1972; WYATT etal.,1972;HAYES etal.,1976;HAYES etal.,1977).The neurotoxic actionofpenitremA inmiceandratsismostunusualbutremainsunexplained (WILSON etal.,1972).The effects ofselected drugson penitremA-inducedtremorsinmiceand ratssuggestthatthe toxinmay actcentrally byinhibition ofinhibitory interneurons (STERN, 1971).WILSON etal.

Antiprotozoal activity in citrinin

SummaryCitrinin has an inhibitory effect on growth ofT. pyriformis. Citrinin also caused a shift to smaller cell size, particularly at the higher concentrations (25, 50 and 100 μg/m1). The mycotoxin exerted only a marginal effect on the respiration of T.pyriformis; but citrinin (25 μg/m1) induced an inhibitory effect on DNA, RNA and protein content. The greatest decrease was in RNA, while smaller decreases in protein and DNA were observed. A bioassay employing T.pyriformis was determined; the lower limit of citrinin detection was between 1 and 5 μg/m1.