William W. Carlton

Response of mice to the chelating agents sodium diethyldithiocarbamate, alpha-benzoinoxime, and biscyclohexanone oxaldihydrazone.

Abstract Weanling albino male mice were fed for 7 weeks the chelators diethyldithiocarbamate, α-benzoinoxime, and biscyclohexanone oxaldihydrazone (cuprizone) mixed with chicken mash containing 10% lard. Diethyldithiocarbamate or α-benzoinoxime at 0.1% and 0.5% of the diet did not produce clinical signs of intoxication. Growth was not affected; gross and microscopic lesions were not observed in several tissues examined. Growth, reduced slightly by 0.1% cuprizone, was severely depressed by a level of 0.5%. Clinical signs were restricted to paresis in the latter group. Microscopic lesions present at both levels of cuprizone feeding were confined to the central nervous system and consisted of edema, most prominent in the cerebellar cortex, and a noninflammatory demyelination. Edema was, however, also observed in the cerebellar medulla and midbrain. Hydrocephalus and cerebral edema occurred in mice fed cuprizone at a level of 0.5% of the diet. Supplementation of the 0.5% cuprizone diet with 25, 50, or 100 ppm copper did not alter significantly the neural lesions induced by the chelator. Only cuprizone feeding interrupted pregnancy. Pregnant mice fed 0.5% and 0.3% cuprizone from the third day of pregnancy failed to deliver young. The former level resulted in a high percentage of nonviable young when feeding was started on the ninth day of gestation. A level of 0.1% cuprizone did not affect pregnancy when fed from the third or ninth day of gestation. A copper supplement of 130 ppm increased the number of viable young when added to the 0.5% cuprizone diet and fed from the ninth day of gestation, but no young were produced when feeding began on the third day of gestation.

Histopathologic lesions in ducklings caused by Aspergillus flavus cultures, culture extracts, and crystalline aflatoxins☆☆☆

The aflatoxins, metabolites produced by some strains of the fungus Aspergillus flavus, are toxic to many animal species. The present report describes the histopathologic lesions induced in ducklings by feeding or repeated oral administration of cultures of the fungus, culture extracts, and pure aflatoxins B1, B2, and G1. The basic pathologic lesions associated with administration of the compounds at any stage of purity consists of hepatic parenchyma necrosis and proliferation of bile ductule cells. Variations occur only in degree and intensity, the severity of damage increasing with successive purification of the toxic compounds. When administered in 5 daily doses, 2.0 μg of aflatoxin B1 initiated clearly defined bile duct hyperplasia. Lesions of similar severity were caused by 15.6 μg aflatoxin and G1 and 50.0 μg aflatoxin B2, indicating lower biological potency of the latter compounds in this subacute assay.

Hepatomas in Rats and Hepatorenal Injury in Ducklings Fed Peanut Meal or Aspergillus flavus Extract

When rats were fed peanut meal (made from peanuts grown and processed in the United States) for 10–12 months, a high incidence of hepatomas and renal damage was observed. Direct extension of hepatomas to the mesentery and metastasis to the lungs occurred in several animals. Parathyroid adenomas were also seen. Ducklings fed the toxic peanut meal for 4 weeks in short term experiments developed hepatic cell damage and bile duct hyperplasia. Lesions induced in ducklings by intubation with extracts of cultures of Aspergillus flavus for 5 days were comparable to those induced with toxic peanut meal.

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 .

Investigations of dose, age, and duration of administration on the hepatorenal damage induced in mice by cultural products of Penicillium viridicatum

Abstract The following studies were completed in mice fed rice cultures of P. viridicatum : the approximate LD50-14 days, age susceptibility, life span, and pathogenesis of hepatorenal damage. Mice were fed the dried, ground rice culture mixed with a purified diet at concentrations from 2.5 to 30%. Diets producing mortalities closest to 50% were those containing 10 and 15% concentrations of the rice culture. In the age susceptibility study, mice of ages 2–18 wk were fed a diet containing 50% rice culture of P. viridicatum . Although mortality was not great in groups older than 6 wk of age, weight loss, and lowering of packed cell volume occurred in all groups fed the fungal diets. Older mice did not become resistant to the toxic effects of P. viridicatum because hepatic and renal lesions were observed in mice of ages 8, 10, 14, and 18 wk. These hepatic lesions were centered in the biliary system and included necrotizing cholangitis, bile duct proliferation, periductal fibrosis, and ductal ectasia. Hepatic cell changes included marked variation in size and shape of nuclei, bizarre mitotic figures, and multinucleated cells. To provide data on the life span of test mice, mice were fed rice cultures at a 7.5% concentration in the diet. Clinical signs of toxicity included anemia, jaundice, and high mortality. None survived beyond 9 wk of feeding. Most of the mice had hepatic focal necrosis and bile duct proliferation. Tubular necrosis was present in most of the kidneys. The development of the hepatorenal lesions was followed in mice fed 5% P. viridicatum diets and killed after 1–24 wk of feeding. Most of the mice presented signs of toxicity such as roughened hair coats early and many were anemic after 7 wk of feeding. Body weights of test groups were always less than controls. Hepatic changes progressed from perilobular, hydropic, and fatty degeneration of hepatocytes to necrotizing cholangitis followed by hepatic cell necrosis. In more chronically fed mice, proliferation of bile duct cells was accompanied by bile duct hyperplasia and periductal fibrosis, and these changes were followed by bile duct ectasia with loss of hepatic parenchyma. The nuclear alterations in hepatocytes were similar to those described in mice of the age susceptibility study.

Cuprizone toxicosis in mice—Attempts to antidote the toxicity

Abstract Studies were undertaken to evaluate the effects of glutamic acid, glutamine, arginine, pyridoxine, γ-aminobutyric acid (GABA) and glucose on the cuprizone-induced toxicosis and encephalopathy in Swiss albino mice. Cuprizone was fed for 8 wk at dietary concentrations of 0.3, 0.5 and 0.75%. Pyridoxine, amino acids and glucose were fed at 10 times the respective cuprizone concentration. Toxicologic and pathologic abnormalities were not altered by glutamine, arginine or glucose supplementation. Glutamic acid at high dietary concentrations decreased mortality but did not reduce the incidence or severity of the neural lesions. Pyridoxine and γ-aminobutyric acid were protective against certain toxicologic and pathologic effects, reducing mortality and decreasing the incidence and severity of the neural lesions. A working hypothesis of the pathogenetic mechanisms of the cuprizone-induced encephalopathy could include the inhibition of the GABA shunt.

Neural lesions induced in ducklings by isonicotinic acid hydrazide and semicarbazide hydrochloride

Abstract White Pekin ducks fed 0.1% isonicotinic acid hydrazide (INAH) or 0.1% semicarbazide hydrochloride (SCH) showed, after about 2 weeks of feeding, signs of motor disturbances. These included tremors, ataxia and paresis. Microscopic lesions included degenerative changes in the nuclei of cerebellar Purkinje cells, motor neurons of the ventral horn of the spinal cord, and neurons of the medulla oblongata. An additional lesion in INAH-fed ducks was a non-inflammatory demyelination. It was most severe in the cerebellar medulla but was also observed in the dorsal funiculi of the spinal cord and in the optic lobes. Supplementation of the SCH diet with dl -glyceraldehyde (0.5%) or salicylaldehyde (0.5%) failed to prevent the neural lesions. dl -Thyroxine was found to be extremely toxic and had no protective activity against SCH- or INAH-induced changes in the brain and spinal cord. Thiouracil as 0.2% of the diet did not prevent the lesions induced by SCH or INAH feedings and appeared to enhance their toxicity as there was greater weight reduction and increased mortality in thiouracil-supplemented groups.

Pathology of dimethylnitrosamine poisoning in Pekin ducklings.

Summary Pekin ducklings were fed a purified diet containing dimethylnitrosamine at levels of 0.02% and 0.0175%. The level of 0.02% was acutely toxic, weight gains were significantly reduced, and feeding was discontinued after 3 weeks. Abdominal distention was observed in some ducks fed 0.0175% dimethylnitrosamine for 4 weeks. A nonhemorrhagic ascites and hydropericardium were observed at necropsy in both test groups. The livers were atrophic and gray in color. The spleens of ducks fed for 4 weeks were markedly enlarged. Histopathologic lesions in the liver included necrosis of hepatocytes with condensation of the reticulum, bile duct proliferation, bile ductule cell hyperplasia, and thickening of the liver capsule with an increase in reticular fibers. Lymphoid hyperplasia was especially prominent in the ducks fed dimethylnitrosamine for 3 weeks. The lymphocytes were present as nodules and as a diffuse infiltration of cells around radicles of the portal vein. Degeneration and necrosis was observed in the lymphoid tissue of ducks fed 4 weeks. Hyalinization of renal glomeruli was prominent in all ducks, and the hyaline material was strongly PAS-positive but was negative to Congo Red, Oil Red O, and the Ziehl-Nielsen stain. Enlargement of the spleen was restricted to ducks fed 4 weeks. Much of the red pulp of the spleen was replaced by large pale cells with vacuolated granular cytoplasm. This change was considered to be hyperplasia of the reticuloendothelial components of the spleen.

The influence of some cultural conditions on toxigenicity of Penicillium viridicatum

Abstract These investigations were made to devise a suitable culture medium for the production of toxin(s) of Penicillium viridicatum . Also, the manner of culture, shaker or quiet, was evaluated. Penicillium viridicatum was grown in corn steep dextrose medium (CSD), potato dextrose broth (PDB), and a modified Czapek-solution (CPX) with or without sucrose, glucose, yeast extract, casein hydrolyzate, cystine, methionine, casamino acids, or microelements. The cultures were incubated at 23°C for 1 or 2 wk. After incubation the fungus was treated with chloroform, dried at 40°C, ground, and mixed either in a ground duck ration or a purified diet. The fungus and its culture liquor were tested for toxigenicity by feeding to mice. The culture liquor was not toxic. The fungus grown under shake was much less toxic than when grown under quiet conditions. Cultures grown on CSD, PDB, and CPX supplemented with either corn steep, casein hydrolyzate, casamino acids, or methionine and cystine were consistently very toxic, causing high mortality and inducing hepatic and to some extent renal damage. CPX with 1% corn steep and 3% glucose appeared to be the superior medium from the standpoint of fungal yield and toxigenicity.

Comparative pathologie changes in ochratoxicosis and citrinin toxicosis

Ochratoxin A and citrinin are nephrotoxic secondary metabolites produced by species of the genera Aspergillus and Penicillium. Chemically, the ochratoxins are isocoumarin compounds linked to L-β-phenylalanine. Ochratoxin A contains chlorine, ochratoxin B is the dechloro analog and ochratoxin C is the ethyl ester of ochratoxin A. Ochratoxin A is the major metabolite both in respect to occurrence and toxicity [3, 4]. Citrinin exists in the solid state as a P-quinone methide and has been found as a natural contaminant of grains [46]. It has co-occurred with ochratoxin A and was found in feedstuffs associated with field outbreaks of endemic porcine nephropathy [25].