Comparison of Irradiation and Chemical Fumigation Used in Grain Disinfestation on Production of Ochratoxin A by Aspergillus alutaceus in Treated Barley.

Effects of irradiation and fumigation on ochratoxin A production by Aspergillus alutaceus in barley were compared. Ochratoxin A production was enhanced relative to untreated controls, if the toxigenic fungus was inoculated into the grain following irradiation or fumigation with either methyl bromide or phosphine. Ochratoxin A production was diminished, if the toxigenic fungus was inoculated into the grain prior to treatment by irradiation or fumigation. The results are consistent with the interpretation that the perturbative effects of irradiation and fumigation on ochratoxin A production by A. alutaceus in this system are very similar and are mediated by a common mechanism. This mechanism involves disturbance of the competition between the toxigenic inoculum and the endogenous microflora of the grain. Reduction or elimination of the competitive microflora, by whatever treatment, allows production of greater amounts of toxin than would occur in the presence of effective competition. These findings suggest that the potential hazard associated with the possible enhancement of ochratoxin A (and probably other mycotoxin) production in treated grain is similar for irradiation or fumigation treatment. Fumigation for purposes of insect disinfestation of grain has been used for decades, with no evidence of any significant hazard vis-a-vis enhanced mycotoxin production.

Variants of Aspergillus alutaceus var. alutaceus (formerly Aspergillus ochraceus) with altered ochratoxin A production.

The present studies, using Aspergillus alutaceus var. alutaceus Berkeley et Curtis (formerly A. ochraceus Wilhelm) NRRL 3174 along with three other wild-type strains, were undertaken in an attempt to understand the effects of irradiation and other treatments on mycotoxin production in grain. Bedford barley was inoculated with spores of NRRL 3174, gamma irradiated, and incubated at 28 degrees C and 25% moisture. After 10 days of incubation, two colony types, ochre (parental) and yellow (variant), were isolated from the grain. Further culturing of the yellow variant resulted in the spontaneous appearance of a white variant that exhibited greatly enhanced fluorescence under UV light. In subsequent work, we have also isolated variants producing a soluble red pigment. In addition, in model experiments involving irradiation (1 kGy) of pure cultures, induction frequencies ranging between 2 and 4% (survival basis) were observed for the yellow and red variants. Inoculation of these variants into wheat and incubation for 14 days at 28 degrees C and 32% moisture resulted in ochratoxin A production in the relative amounts of 0.09:1:4.6:9.3 for the red, ochre (parental), yellow, and white variants, respectively. Additional characteristics of these isolates are described. Confirmation that the white high-ochratoxin-A-producing variants were derived from the parental strain was demonstrated by obtaining revertant sectors in monoclonal cultures of the variants.

Role of the competitive microbial flora in the radiation-induced enhancement of ochratoxin production by Aspergillus alutaceus var. alutaceus NRRL 3174.

The radiation sensitivity and the toxigenic potential of conidiospores of the fungus Aspergillus alutaceus var. alutaceus were determined after irradiation with 60Co gamma rays and high-energy electrons. Over the pH range of 3.6 to 8.8, the doses required for a 1 log10 reduction in viability based on the exponential portion of the survival curve ranged from 0.21 to 0.22 kGy, with extrapolation numbers (extrapolation of the exponential portion of the survival curve to zero dose) of 1.01 to 1.33, for electron irradiation, and from 0.24 to 0.27 kGy, with extrapolation numbers of 2.26 to 5.13, for gamma irradiation. Nonsterile barley that was inoculated with conidia of the fungus and then irradiated with either electrons or gamma rays and incubated for prolonged periods at 28 degrees C and at a moisture content of 25% produced less ochratoxin A with increasing doses of radiation. Inoculation of barley following irradiation resulted in enhanced ochratoxin levels compared with unirradiated controls. In these experiments, inoculation with 10(2) spores per g produced greater radiation-induced enhancement than inoculation with 10(5) spores per g. There was no radiation-induced enhancement when the barley was surface sterilized by chemical means prior to irradiation. These results are consistent with the hypothesis that a reduction in the competing microbial flora by irradiation is responsible for the enhanced mycotoxin production observed when nonsterile barley is inoculated with the toxigenic fungus A. alutaceus var. alutaceus after irradiation.

Enrichment of progenitor cells from human bone marrow by flow cytometry.

Mononuclear cells, harvested from fresh human bone marrow specimens by density gradient separation, were suspended in phosphate buffered saline and analyzed by flow cytometry in terms of the forward and right angle scattering of the incident light. The rectilinear distribution, obtained by plotting the intensity of light scattered in the forward and right angle directions, contained three regions of interest in which the percentage of cells (Mean +/- standard deviation) with respect to the total was as follows: Region 1: 17.6 +/- 9.9; region 2: 5.3 +/- 1.4; region 3: 71.7 +/- 9.4. Cells from each region were sorted by flow cytometry and plated in semi-solid agar containing cell conditioned medium supportive of myeloid colony formation. Cells from region 2 contained the majority of progenitor cells that gave rise to such colonies at a plating efficiency that rose in proportion to the extent by which the region 2 cells in samples was increased through sorting. This increase in plating efficiency was 6 to 43 fold. Thus, region 2 of the cytometric distribution of cells from normal, unstained human bone marrows was a good source of myeloid progenitor cells.

Response of human bone marrow progenitor cells to X-rays in vitro.

In 25 out of 33 cases the survival response of myeloid progenitor cells from fresh human bone marrows, X-irradiated in vitro in phosphate-buffered saline at 23 degrees C, was characterized by a two-term exponential relationship, with D0 values of 0.42 +/- 0.19 Gy and 1.38 +/- 0.37 Gy, respectively. In the remaining eight cases for which the colony-forming efficiency was 10 times lower, survival followed a single exponential function with D0 = 1.18 +/- 0.15 Gy. The biphasic response at 23 degrees C became a single exponential response when the temperature at irradiation was 30 or 37 degrees C.

In vitro response of human bone marrow progenitor cells to superoxide radicals.

Human bone marrow progenitor cells, grown on solid agar, developed colonies of the white blood cell series, principally granulocytes, monocytes and macrophages. Following exposure to superoxide radicals, generated photochemically, a fraction of the progenitor cells was inactivated as evidenced by loss of colony formation. The loss in proliferative capacity depended on the illumination time and individual nature of the donated specimens. Protection of the progenitor cells by active superoxide dismutase depended on the illumination time and temperature. Inactive superoxide dismutase and active catalase did not protect the cells from the photochemical inactivation process.

Radioprotection by superoxide dismutase of macrophage progenitor cells from mouse bone marrow.

X-ray survival of cultured macrophage progenitor cells from mouse bone marrow was represented by a two-component curve, both in the absence and presence of superoxide dismutase. Protection by the enzyme was limited to the radiosensitive fraction, for which a dose modifying factor of 2.8 +/- 0.7 was obtained. Catalase did not protect. Survival of the radiosensitive fraction, with and without exogenous superoxide dismutase, was temperature-dependent, whereas that of the radioresistant fraction was not. In the former case, the energy required for the enzyme-treated cells was approximately 13kJ/mol.

Identifying superoxide-sensitive progenitor cells in mouse bone marrow.

Macrophage progenitor cells in bone marrow, that develop into attached colonies in liquid culture medium, contain a fraction of cells sensitive to photochemically generated superoxide radicals. This fraction varies from one animal to another. Populations of cells containing the superoxide-sensitive fraction show a greater sensitivity to X-rays than do populations in which this fraction has been photochemically inactivated. The change in radiosensitivity was proportional to the superoxide-sensitive fraction.

Concentration-dependent inactivation of superoxide dismutase.

The inactivation yield of superoxide dismutase (superoxide: superoxide oxidoreductase, EC 1.15.1.1) from bovine erythrocytes, when Co60-gamma-irradiated in air, N2 or N2O-saturated solutions, increases exponentially with the initial enzyme concentration. In aerated solutions at less than or equal to 10 micrometers, the inactivation process continues in a concentration-dependent manner in the subsequent 72 h. This post-irradiation effect is inhibited by catalase. Above 10 micrometers, radiation-induced inactivation of the enzyme is partially reversed in a concentration-dependent manner and is not affected by catalase. In aerated and N2O-saturated solutions, competitive scavenging of radiation chemical species by catalase and EDTA in combination reduces the inactivation yield by 80%; the residual yield remains dependent on enzyme concentration. Radiation-induced loss of copper and zinc initially exhibits a linear dose-response relationship and is less severe than the drop in enzyme activity.

Tissue distribution of bovine 125I-superoxide dismutase in mice.

Intravenously injected 125I-bovine superoxide dismutase in mice is essentially cleared from organs in 24 h. The 125I activity per g of tissue at 1 h varies among organs with the brain and kidneys showing the lowest and highest levels, respectively. Within tissues, degradation occurs so that, at 1 h after administration, 5-74% of the 125I activity remains associated with the enzyme, depending on the tissue. A small amount of the labelled enzyme is found in washed cells of thymus, lung, spleen, liver and brain.

Radioprotective effect of superoxide dismutase on model phospholipid membranes.

1. Hydroperoxide formation in model membranes was measured via the net increase in absorbance at 232 nm after exposure to X-rays or 137Cs gamma rays in the presence and absence of bovine superoxide dismutase and other radical scavengers. 2. Membranes X-irradiated in air to 4200 rad at 210 rad/min exhibited a large increase in absorbance, a major portion of which was O2--mediated since active superoxide dismutase at 1 mug/ml reduced it by more than 80% to the level observed in N2O. In N2 the change in absorbance was smaller than in N2O but not in proportion to the halving in OH production. 3. The net absorbance of membranes exposed to a constant dose from 137Cs increased with decreasing dose rate. A minor component of this effect was due to exposure protraction with decreasing dose rates while the major component was attributed to long chain reactions initiated by ionizing radiation. A corollary effect was also observed, namely, that with reducing dose rate the dose required to elicit a constant absorbance change decreased. Both aspects were abolished by superoxide dismutase at 1 mug/ml. 4. The enzyme protected membranes after an acute exposure and from low level radiation at natural background while its inactivated form sensitized.