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Biomedical subjects

E B Lillehoj

Publications and source records attributed to E B Lillehoj.

At least 19 recordsLinked to original sources

Relationship of byssinosis to the generation of oxygen radicals by bract tissues of cotton plants.

Byssinosis is a hazardous respiratory disorder of workers in natural fiber processing industries and, in the case of cotton, is caused by exposure to respirable dust generated from leafy trash associated with raw fibers. To understand the chemical characteristics of involucral trash components that might contribute to bysinosis, we examined the human airway constricting activity and oxygen radical generating activity of dry, frost-killed cotton bracts. In response to inhalation of aerosolized bract extracts, the expiratory flow rates of human volunteers at 40% of vital capacity during partial forced expiration decreased by 3 to 32%. These values enabled us to identify two potentially byssinogenically active bract specimens, a specimen virtually inactive, and a fourth intermediately so. Using spin trapping techniques of electron spin resonance spectrometry, we found that all specimens catalyzed the generation of hydroxyl (preponderantly) and superoxide radicals from hydrogen peroxide. However, the weakest constrictor was the most potent catalyst, and vice versa. This was consistent with transition metal content of the specimens; the most potent catalyst also contained the largest amounts of those metals, suggesting a Fenton-type reaction mechanisms. Other possibilities for the inverse relationship of airway constricting (byssinogenic) activity with oxygen radical generation are discussed. We also found that neither aflatoxin nor endotoxin, contingent contaminants of bracts, catalyzed oxygen radical production from hydrogen peroxide.

Aflatoxin B1

Enzymes in aflatoxin 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 of Aspergillus 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 enzyme's amino acid composition determined. A cDNA library has been constructed from mRNA isolated from Aspergillus parasiticus mycelia during the onset of AFB1 biosynthesis for the purpose of identifying the genes responsible for aflatoxin biosynthesis.

Aflatoxin B1

Preharvest aflatoxin contamination: effect of moisture and substrate variation in developing cottonseed and corn kernels.

Variations in moisture and substrate in preharvest corn kernels and cottonseed were linked with the ability of Aspergillus parasiticus to infect the seed and produce aflatoxin. Osmotic pressures and moisture content (MC) levels of developing starch-rich corn kernels and lipid-rich cottonseed were determined. For in vivo studies, corn kernels and cottonseed were inoculated with A. parasiticus conidia and retained on plants through maturation. For in vitro studies, samples of corn kernels and cottonseed were collected at various stages, sterilized, inoculated, incubated for 2 weeks, and assayed for toxin. Aflatoxin levels were highest in corn kernels inoculated at 28 days postflowering (52% MC) in both the in vivo and in vitro tests. Toxin concentrations in cottonseed were greatest with inoculation at 35 days postflowering (70% MC) in seed retained on the plant, but toxin accumulation continued to increase with the maturity of the seed inoculated in cottonseed used in the in vitro trials. Moisture and substrate conditions in the midrange of seed development provided optimum conditions for fungal development and toxin production in seed retained on the plant.

Aflatoxins

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.

Aflatoxins

Ochratoxin A carcinogenesis in the (C57BL/6J X C3H)F1 mouse.

The potential carcinogenic effects of the mycotoxin ochratoxin A [(OA); CAS: 303-47-9] were assessed in a 24-month feeding study in male and female (C57BL/6J X C3H)F1 (B6C3F1) mice. The mice were assigned to 3 groups of 50 males and 50 females each; group 1 mice were the controls, group 2 mice were fed 1 ppm OA, and group 3 mice were fed 40 ppm OA. Renal neoplasms, both carcinomas and adenomas, were found only in male mice of the 40-ppm dose group. Fourteen of 49 animals that survived at least 20 months had neoplasms morphologically consistent with renal carcinoma. Renal adenomas were present in some of these mice and in other 40-ppm-group males, making a total of 26 mice with renal adenomas. All male mice of the 40-ppm dose group had nephropathy characterized by varying degrees of renal tubular dilation, attenuation and hyperplasia of lining epithelium, and proliferation of regenerative tubules. Females of the 40-ppm dose group had similar but less severe renal changes but no carcinomas or adenomas. Compound-related renal lesions were absent in the 1-ppm dose group. The incidence of hepatocellular neoplasms was slightly increased in male and female mice fed diets containing OA. These results indicate that OA is a renal carcinogen in male B6C3F1 mice and a hepatic carcinogen in female mice of this strain.

Animals

Aflatoxin contamination of maize kernels before harvest. Interaction of Aspergillus flavus spores, corn earworm larvae and fungicide applications.

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.

Aflatoxins

Simple method for isolation of 4-deoxynivalenol from rice inoculated with Fusarium graminearum.

A new method for preparative isolation of 4-deoxynivalenol (DON) is presented. This method avoids the loss of material during purification on silica gel by column chromatography. DON and 3-acetyldeoxynivalenol in crude extracts of rice inoculated with Fusarium graminearum were converted to triacetyldeoxynivalenol; the acetylated product was easier to purify by silica gel chromatography than DON is. After hydrolysis and further purification on a charcoal-alumina column, the 71% pure DON was recovered in yields as high as 450 mg of DON per kg of rice. Subsequent separation on a Sephadex LH20 column yielded DON that was greater than 90% pure.

Chromatography, High Pressure Liquid

Variability of bright, greenish-yellow fluorescent particles and aflatoxin in ground blends of Zea mays.

Developing corn (Zea mays) kernels of single cross B73 X Mo17 grown near Columbia, MO, were inoculated with conidia of Aspergillus flavus or A. parasiticus. After harvest bright greenish-yellow fluorescing (BGYF) kernels were selected under ultraviolet black light (365 nm); they contained 3696 ng/g (A. parasiticus strain NRRL 3145) and 6666 ng/g (A. flavus strain UGA 5337) aflatoxins, respectively. The BGYF-free kernels were obtained from the same hybrid grown in northern Iowa. No BGYF kernels nor aflatoxins were found in 10 assays. To assess the variability in detection of BGYF particles and aflatoxin in ground corn blends, a series was developed by mixing BGYF kernels with BGYF-free kernels in various ratios. In the finely ground samples, standard deviations decreased significantly (P = 0.05) among blends for aflatoxin values in mixtures with higher levels of BGYF particles. Strain 3145 (A. parasiticus) was significantly (P = 0.01) different from strain 5337 (A. flavus) for aflatoxins concentration and the number of grams of BGYF in the blends (r greater than 0.99). The percent fines (passed a 40-mesh screen) in ground, 100% BGYF kernels was significantly (P = 0.01) higher for strain 3145 (A. parasiticus) inoculated samples than for strain 5337 (A. flavus) in spite of equivalent grinding procedures. This observation showed that the relationship between aflatoxin levels determined for a sample and the fraction of BGYF material in the blend was dependent on the fungal-strain source of the BGYF kernels used in the blend.

Aflatoxins

Occurrence of ochratoxin- and citrinin-producing fungi on developing Danish barley grain.

The distribution of Aspergillus and Penicillium spp. isolates that produce ochratoxin A (AO) and citrinin was studied on developing barley from diverse locations in Denmark during the 1977 crop year. From 33 test fields, 243 isolates of the two genera were obtained with significant numbers of isolates on the grain at the earliest sampling date (1-2 weeks postanthesis). No specific argronomic factor was linked to the distribution of Aspergillus and Penicillium spp. A taxonomic study of 108 representative isolates of the two genera showed that A. flavus, P. chrysogenum, and P. purpurrescens were the predominant species. An investigation of the toxin-production capability of the identified species showed that seven strains (6.5%) produced OA and a single isolate synthesized citrinin. P. purpurrescens was the dominant OA-producing species with four toxin-positive isolates.

Aspergillus

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, jejunum, ileum, and cecum exhibited the greatest counts at 18 hr after toxin exposure.

Animals

The fate of aflatoxin in naturally contaminated corn during the ethanol fermentation.

Corn naturally contaminated with aflatoxin was used as a substrate in the ethanol fermentation. Distribution of toxin in several process and recovery fractions was identified. Although little degradation of the mycotoxin occurred during fermentation, no toxin appeared in the distilled alcohol. As accumulation of toxin in spent grains represents a potential problem in use of the material as animal feed, several decontamination procedures were tested. Sodium hydroxide, ammonium hydroxide, sodium hypochlorite, and hydrogen peroxide were identified as efficient agents of toxin degradation.

Aflatoxins

Aflatoxin variation among corn samples with varying ratios of Aspergillus flavus-inoculated/noninoculated kernels.

Aflatoxin levels and physical properties of corn kernels inoculated with Aspergillus flavus during development and noninoculated kernels were compared in samples with various proportions of the 2 kernel types. The relationship between mean toxin levels and associated standard deviations of 5 samples demonstrated a linear association from the lowest toxin in noninoculated corn through a mixture of 60% inoculated/40% noninoculated. However, at the highest toxin level in the 100% inoculated material, a reduction in sample variation was observed. Examination of individual kernal weights showed that inoculated kernels were distinctly lighter than noninoculated seed. A uniform grinding procedure of the samples yielded heterogeneous particle sizes based on the starting corn. The large particle fraction (greater than 500 micrometers) decreased from 100% noninoculated kernels through the mixtures to the 100% inoculated seed; particles below 150 micrometers were most abundant in the ground samples from inoculated kernels. In addition, the density of particles within a size category varied; lower densities were observed in samples obtained from A. flavus-inoculated kernels.

Aflatoxins

Bioproduction of [14C]ochratoxin A in submerged culture.

A number of Aspergillus and Penicillium species were tested for production of ochratoxin A (OA) in several media. After 8 days of static incubations of submerged cultures at 28 degrees C, toxin yields of 25 and 30 micrograms/ml were obtained with Aspergillus alliaceus NRRL 4181 in Ferreirás and 2% yeast extract-4% sucrose media, respectively. However, the largest production observed in the preliminary screening was 54 micrograms/ml; this highest level was produced by A. sulphureus NRRL 4077 in a modified Czapek solution. The medium contained the basal salts and sucrose of Czapek plus urea (3%) and corn steep liquor (0.5% solids). A time study of toxin production demonstrated maximum yield of 350 micrograms/ml by the A. sulphureus isolate in the modified Czapek medium after 11 days of static incubation at 28 degrees C. The optimal production conditions were employed in additional tests designed to measure the efficiency of 14C incorporation from sodium [1-14C]-acetate into OA. Samples (20 microCi) of sodium acetate were added to separate culture flasks at 24-h intervals during the initial 9 days of the fermentation. Addition of [14C]acetate on day 4 of incubation provided the maximum yield of labeled OA. The highest specific activity of labeled toxin obtained was 0.07 microCi/mg of OA and the maximum incorporation rate of labeled acetate was 5.3%.

Acetates

Complement activity, serum protein, and hepatic changes in guinea pigs given sterigmatocystin or aflatoxin, alone or in combination.

Effects of either sterigmatocystin or aflatoxin, alone or in combination, given orally to guinea pigs were studied. Sterigmatocystin and aflatoxin B1 given alone and in combination at 4.2 mg/day and 0.01 mg/day, respectively, markedly reduced body weight. Although changes in total serum protein were not marked in any of the guinea pigs in this study, sterigmatocystin given alone and aflatoxin given alone significantly ( less than 0.05) decreased alpha2-globulin. The combination of toxins significantly (P less than 0.01) increased albumin and significantly (P less than 0.01) decreased both alpha2- and beta-globulins. Sterigmatocystin depressed complement activity, although not significantly. However, the combination of sterimatocystin with 0.01 mg of aflatoxin B1/day (an amount that does not affect complement activity alone) significantly (P less than 0.01) reduced complement activity. Increased severity of lesions was not found in guinea pigs given aflatoxin at 0.01 mg of B1 equivalents/day in addition to the sterigmatocystin.

Aflatoxins

Aspergilus flavus and aflatoxin in Iowa corn before harvest.

Aspergillus flavus and aflatoxin were detected in ears of Iowa corn on plants before harvest in 1975. Presence of the fungus was associated with kernel injury caused by the second generation European corn borer. Amounts of aflatoxin B1 in corn from a limited number of selected ears ranged from 1 part per billion to 1560 parts per billion with a mean of 430 parts per billion.

Aflatoxins

Experimental porcine nephropathy: changes of renal function and structure perorally induced by crystalline ochratoxin A.

Nine pigs were fed crystalline ochratoxin A in amounts corresponding to a feed level of 1 mg per kg for 3 months. The only observable lesion developed was a kidney damage, identical to the naturally occurring porcine nephropathy. The changes of renal function was characterized by impairment of proximal tubular function, indicated by a decrease of the ratio TmPAH/CIn, of the ability to concentrate urine, and by an increased urinary excretion of glucose. The decrease of the ratio TmPAH/CIn is correlated with time of exposure to ochratoxin A. The changes of renal structure were characterized by degeneration of the proximal tubules, leading to tubular atrophy accompanied by interstitial fibrosis. At the end of the experiment the kidney, liver, adipose and muscular tissue of the slaughtered pigs contained sizable amounts of ochratoxin A residues. As the pigs would have passed the meat inspection this represents a possible health problem. The changes observed in this study are identical to those observed by feeding to pigs grains naturally contaminated with ochratoxin A.

Adipose Tissue