Michael E. Stack

Myelotoxicity and macrophage alteration in mice exposed to ochratoxin A

Six- to seven-week-old female B6C3F1 mice were administered a total of 0, 20, 40, or 80 mg/kg of ochratoxin A (OCT A) ip on alternate days over an 8-day period. Twenty-four hours following the final dose, histopathology, bone marrow, and macrophage parameters were assayed. There was a dramatic dose related decrease in thymic mass with the mean thymus weight of the high dose animals being only 33% of controls. Histologic evidence of nephrotoxicity was minimal and restricted to the inner cortex. Myelotoxicity was present as evidenced by bone marrow hypocellularity, decreased marrow pluripotent stem cells (CFU-S), granulocyte-macrophage progenitors (CFU-GMs), and decreased 59Fe uptake in marrows and spleens of exposed mice. Peritoneal macrophages from sc as well as ip injected mice demonstrated increased phagocytic capacities and increased capacity to inhibit tumor cell growth. These alterations in bone marrow cells and macrophages suggest myelotoxicity is an additional potential hazard of OCT A exposure.

Production of Alternariol and Alternariol Methyl Ether by Alternaria alternata Grown on Fruits at Various Temperatures

Two toxigenic strains of the fungus Alternaria alternata (ATCC 56836 and ATCC 66868) were grown on surface-disinfected, fresh, ripe fruits and tested for the production of alternariol (AOH) and alternariol methyl ether (AME). Examined fruits included strawberries; red and green seedless grapes; concord grapes; red delicious, golden delicious, and gala apples; and blueberries. After inoculation, fruits were incubated at 4, 10 degrees C, or room temperature (approximately 21 degrees C) for up to 3 weeks. At weekly intervals, duplicate samples were analyzed for AOH and AME by using liquid chromatography. Results indicated that A. alternata and its metabolites were not a major problem in strawberries due to the presence of fast-growing molds like Rhizopus and Botrytis that outgrew and possibly inhibited Alternaria. Both Alternaria strains showed limited growth on apples, although fast-growing molds were not present after surface disinfection; AOH and AME were produced only by the ATCC 56836 strain on the golden delicious and gala varieties, (ranging from <0.1 to 5 microg/g and <0.1 to 14 microg/g for AOH and AME, respectively). Restricted growth of both strains without toxin production occurred in blueberries, whereas moderate growth and AOH (<0.1 to 3,336 microg/g) and AME (<0.1 to 1,716 microg/g) production took place in grapes.

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.

Species- and sex-specific renal cytotoxicity of Ochratoxin A and B in vitro

Four different cell models were chosen for comparison of OTA and OTB toxicity: primary porcine (PKC), rat (RPTC) and human renal proximal epithelial cells (HKC) from both sexes and a porcine renal cell line: LLC-PK1. Culture conditions were tested and optimized for each respective cell type (species/sex and origin). All cell types were characterized for epithelial origin and growth patterns and following optimization of dosing strategies and assay procedures, a strict study design was implemented to avoid systemic variations. Due to possible sensitivity differences, three simple endpoints were chosen to provide basic data for interspecies comparison: neutral red uptake, MTT reduction and cell number. Of the endpoints tested neutral red appeared the most sensitive, although all three parameters yielded comparable EC50s. Sex-differences were observed between male and female HKC cells following 96 h exposure to OTA, with HKC(m) being more sensitive than HKC(f). No sex-difference was observed in PKC cells, however, the PKC were approximately 3 and 10 times more sensitive than HKC(m) and HKC(f), respectively, to OTA and OTB. Interestingly, the CI95 of the EC50 values obtained for OTA (15.5-16.5 microM) and OTB (17.0-2 1.0 microM) were comparable in the PKC cells. In contrast, OTB had lower cytotoxicity than OTA in HKC and LLC-PK1 (approx. 2-fold) and no effects in RPTC. Overall, HKC(m) were nearly as sensitive as PKC towards OTA, followed by RPTC, LLC-PK1 and HKC(f), thus suggesting a sex specific sensitivity in humans towards OTA induced cytotoxicity.

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 .

Molds and tenuazonic acid in fresh tomatoes used for catsup production.

The mold flora was determined for 146 samples of fresh but visibly moldy tomatoes collected from sorting belts in tomato catsup processing plants in California and in Midwestern and Eastern United States. Mold found in 141 of the samples included at least 22 genera, principally Alternaria , Aspergillus , Cladosporium , Fusarium and Penicillium , and 51 species. The California tomatoes were dominated by Geotrichum candidum and species of Aspergillus and Penicillium ; Midwest and East tomatoes were dominated by Alternaria . This suggested that the predominant molds in tomatoes may differ, depending on geographical source. Tenuazonic acid (TA), a toxic metabolite of Alternaria spp., was found in 73 of the samples at a range of 0.4 to 69.7 (average 4.94) μg/g of moldy tissue; however, Alternaria spp. were not found in 35 of the 73 TA-positive samples. It is possible that other molds may produce TA or that the toxin-producing Alternaria died off before our sampling.

Isolation and identification of dihydrocitrinone, a urinary metabolite of citrinin in rats

Dihydrocitrinone, 3,4-dihydro-6,8-dihydroxy-3,4,5-trimethylisocoumarin-7-carboxylic acid, was isolated and identified as a urinary metabolite after oral administration of citrinin to rats. Male and female Osborne-Mendel rats received 30 mg citrinin/kg body weight by oral intubation. The metabolite dihydrocitrinone was present in urine collected at 0-2, 2-4, 4-6, 6-8, and 8-24 h after treatment. Only unchanged citrinin was found in blood collected 24 h after administration of the compound. The metabolite had a blue fluorescence and the same Rf on thin-layer chromatography, the same retention time on reverse-phase high-pressure liquid chromatography, and the same mass spectrum as an authentic sample of dihydrocitrinone.

Ochratoxins A and B, Xanthomegnin, Viomellein and Vioxanthin Production by Isolates of Aspergillus ochraceus from Green Coffee Beans

Isolates from Aspergillus ochraceus obtained from green coffee beans were cultured on rice and water. After 20 d of growth the cultures were extracted with chloroform and the extracts were analyzed by high performance liquid chromatography for ochratoxin A (OA), ochratoxin B (OB), xanthomegnin (X), viomellein (V) and vioxanthin (VX). Forty-three percent of the isolates produced OA at an average level of 397 μg of toxin/g rice, 17% produced OB at an average level of 312 μg/g, and 84% produced X, V, and VX at an average level of 281, 417 and 386 μg/g, respectively. The highest levels of toxin production were OA, 2088 μg/g; OB, 3375 μg/g; X, 1562 μg/g; V, 2514 μg/g; and VX, 2054 μg/g. VX has not previously been reported as an A. ochraceus metabolite.

Structures of xanthoviridicatin D and xanthoviridicatin G, metabolites of penicillium viridicatum: application of proton and carbon-13 NMR spectroscopy

The structures of xanthoviridicatin D and xanthoviridicatin G, toxic metabolites of Penicillium viridicatum, have been shown to be 3 and 4a, respectively by spectroscopic studies.