Aflatoxin B₁ (AFB₁,) is a potent liver carcinogen to a number of animal species including rainbow trout (Salmo gairdneri). Microsomal activation is required for the in vitro conversion of AFB, to a reactive metabolite, thought to be the AFB₁,-2,3-oxide, which will bind nucleic acids, produce toxicity, and cause mutagenesis. Nucleic acid-AFB₁ adduct formation is believed to be an indication of cancer initiation. Initial time-course and dosage experiments were conducted to establish fundamental binding data in rainbow trout. Dietary casein and fish protein concentrate (FPC) fed at 40, 50, 60, or 70% in the diet, and cyclopropenoid fatty acids (CPFA), were measured for their effect on in vivo AFB₁, binding to trout liver DNA. Both were previously reported to dramatically alter AFB₁, induced hepatocarcinogenesis. Rainbow trout and coho salmon (Oncorhynchus kisutch), two Salmonid species varying greatly in their sensitivity to AFB₁, were compared for their relative ability to produce AFB₁-DNA adducts. Correlations between trout liver mixed function oxidase (MFO) activity and AFB₁, binding to trout liver DNA were attempted by use of β-napthoflavone, a powerful enzyme inducer in rainbow trout. Binding of AFB₁, to trout liver DNA over the 48 hour(h) period studied reached a maximum value at 24 h. Increasing AFB₁, dosage produced a linear response in AFB₁-DNA adduct formation. Binding was significantly greater in the 70% casein fed fish than in the 70% FPC group. Binding in the 40, 50, and 60% casein fish was non-significantly greater than the corresponding FPC groups. AFB₁, binding to liver DNA was greatest in each source at the 60% protein level. Binding in rainbow trout was at least 20-fold greater by comparison than in coho salmon. CPFA reduced AFB₁, binding to trout liver DNA at each protein diet employed, although non-significantly. Pretreatment of fish with β-napthoflavone reduced the total level of AFB₁ in the liver and AFB₁-DNA adduct formation by 55 and 40%, respectively. Dietary protein and CPFA apparently alter tumor formation through promoter effects since binding was unaffected. The tumor resistance of coho salmon and β-napthoflavone pretreated animals is possibly due to reduced initiation of of the toxic lesion. / Graduation date: 1981

Indole-3-carbinol (I3C) and butylated hydroxyanisole (BHA), anti-carcinogens present in the human diet, were tested for their in vivo and in vitro effect on aflatoxin B₁. (AFB₁) metabolism, and DNA adduct formation in the rainbow trout. Dietary BHA at either 0.3 or 0.03% had no effect on the hepatic tumor incidence of trout exposed to a 0.5 ppm AFB₁ solution as embryos, or when fed prior to and during dietary exposure to 10 ppb AFB₁. Previous studies have shown 0.1% I3C to inhibit AFB₁-induced hepatomas in trout. When fed at 0.2%, I3C produced a 70% reduction in average in vivo hepatic DNA binding of injected AFB₁ over a 21 day period compared to controls. A similar study with 0.3% BHA had no effect on AFB₁-DNA binding over a 7 day period. One hr incubations of AFB₁ with freshly isolated hepatocytes from either BHA-, I3C- or control-fed trout showed no differences in AFB₁ metabolism or DNA binding between BHA hepatocytes and controls. However, I3C hepatocytes had 20% less DNA binding with a 2-fold increase in aflatoxin M₁ production. Additions of 0, 1, 10 or 100 uM BHA or I3C to hepatocytes isolated from trout fed a control diet had no effect on AFB₁-DNA adduct formation except for a 20% decrease in the 100 uM BHA hepatocytes. A 24 hr distribution study of injected [³H]-AFB₁ in trout fed 0.3% I3C showed less total radioactiuity in the blood and liver at all times examined, compared to controls. These reductions were accountable primarily as reduced levels of AFB₁ bound to red blood cell DNA, reduced plasma levels of the metabolite aflatoxicol (AFL), and decreased levels of AFB₁ and polar metabolites in the liver of I3C trout. Total radioactivity was significantly elevated in the bile of I3C fish resulting from a 7-fold increase in aflatoxicol-n. glucuronide levels over controls. AFL glucuronide levels were similar between treatments. Total radioactivity remaining in the carcasses of I3C or control trout was similar. These data indicate that I3C inhibits AFB₁ hepatocarcinogenesis in trout through changes in carcinogen distribution, metabolism and elimination leading to reduced initial DNA damage. BHA does not appear to alter enzymes responsible for AFB₁ metabolism, and though it may have a weak direct affect on AFB₁-DNA adduct formation, this does not appear to be of importance in vivo since BHA had no effect on AFB₁-induced carcinogenesis. / Graduation date: 1986

Selected parameters that affect crude toxin production in both primary culture (growing cell culture) and resting culture were examined during efforts to produce sufficient crude toxin for purification. The parameters included carbohydrate and label source and concentration, cell concentration, incubation time and temperature and shaker speed. A higher yield of crude toxin was obtained with 0.005 M glucose plus 0.002 M acetate than with 0.08 M glucose alone. Acetate-1-¹⁴C gave a higher specific activity of crude toxin than glucose-6-¹⁴C. Maximum yields and incorporation were obtained with washed cells from 72 hour primary culture incubated with nitrogen free replacement medium for 12 hours at 30°C on a rotary shaker at 200 rpm. A number of chromatography supports and solvent systems were examined in an attempt to purify crude toxin. Activated silica gel H eluted from a column with a gradient of chloroform: methanol from a metering pump gave fractions of pure aflatoxin B₁ Comparison of the ratios of the ultraviolet radiation absorbances of the toxin to pure reference aflatoxin B₁ was a criteria for chemical purity. Retention of specific activity after chromatography, hydrogenation to tetrahydrodeoxoaflatoxin B₁ and preparation of the hemiacetal and epimeric acetates indicated the label was incorporated in aflatoxin B₁. A successful method for the preparation of ¹⁴C labeled aflatoxin B₁ from resting cultures of Aspergillus parasiticus ATCC 15517 in a defined synthetic medium containing 0.02 M glucose and 0.005 M acetate is described. After purification ¹⁴C labeled aflatoxin B₁ of specific activity 744 μci per mmole was obtained from 2 mci acetate-1-¹⁴C of specific activity 1 mci per mmole. / Graduation date: 1972

Aflatoxins B₁, Q₁ and aflatoxicol, R [subscript o] F, but not B₂ were activated in the presence of NADPH by the 105,000 x g pellet from rainbow trout (Salmo gairdneri), Mt. Shasta strain, liver to products lethal to Bacillus subtilis GSY 1057 (metB4, hisAl, uvr-1). Electron microscopy confirmed the microsomal fraction primarily contained smooth endoplasmic reticulum. An NADPH mixed function oxidase with neo-tetrazolium reductase and aldrin epoxidase activity was present in the microsonaes. The activity of the oxidase was reduced parallel to a decrease in lethal factor production by piperonyl butoxide, NADPH deprivation, heat treatment and certain diets. After incubation of microsomes with ¹⁴C labeled B₁, the activity was found in unaltered B₁ and three extremely polar metabolites designated NM (3. 6-5. 3%), B [subscript 2a] A (1-5%) and M₁A (< 1%). They were eliminated or reduced along with microbial lethality when cytosine and cysteine were added to the incubation media. The structural requirement for the vinyl ether of B₁, R [subscript o] F and Q₁ and the nature of the enzymatic reaction were consistent with the hypothesis that the conapounds were metabolized to highly reactive and unstable electrophilic products which bound to nucleophiles such as cytosine and were lethal to B. subtilis. Aflatoxicol, R [subscript o] F, was isolated from liver homogenates and was apparently fornaed by an NADPH-dependent soluble enzyme of the 105,000 x g supernatant from rainbow trout. R [subscript o] F and its diastereomer, R [subscript o] R, were prepared by chemical reduction of B₁. Their identity was confirmed by UV and mass spechrometry. The ten-day LD50 value was 0.66 mg/kg for R [subscript o] F compared to 0.46 mg/kg for B₁. No mortality was observed, from R [subscript o] R at equivalent doses. Similar gross and microscopic pathological changes were observed for B₁, R [subscript o] F and R [subscript o] R. The degree of damage paralleled the LD50 values. After eight months of feeding, 20 ppb of B₁ and R [subscript o] F and 36.6 ppb R [subscript o] R gave 56.3%, 26.2% and 0% incidence of hepatoma. Addition of 50 ppm of cyclopropenoid fatty acids increased the incidence to 96.3%, 93.8% and 55% for B₁, R [subscript o] F and R [subscript o] R. It was concluded that aflatoxicol was not an effective means of detoxication of B₁, but instead extended the presence of a potentially toxic and carcinogenic compound. / Graduation date: 1974

The effect of cycling temperatures on production of aflatoxin by Aspergillus flavus (V3734-10) when grown upon various substrates was studied. The parameters of temperature and time were selected to simulate environmental conditions in Oregon during harvest of filberts and walnuts. The heat input required for aflatoxin synthesis in terms of degree hours per day were calculated and may be used as an index to predict potential danger of aflatoxin contamination. Conditions which generated less than 208 hours per day did not receive sufficient heat to induce growth and metabolism. When heat input ranged between 208 and 270 hours per day, growth and metabolism occurred with the development of a yellow pigment. There was not sufficient heat input, however, to induce the idiophase, sporulation and subsequent aflatoxin synthesis. Above 270 hours per day the culture entered the idiophase, sporulation occurred and aflatoxin was produced. The heat requirements for aflatoxin production was compared to the degree hours produced in the orchards by prevailing weather conditions at the time of harvest. The nuts are most susceptible to contamination and fungal growth during harvest when the nuts are damp and on the ground. During October and November, heat input is too low for aflatoxin production. From mid September to October heat input may be sufficient for aflatoxin production but at this time most walnuts and filberts are still tree borne and moisture in the environment is likely to be a limiting factor. A. flavus was found to be a poor competitor when grown with the natural fimgal flora isolated from moldy nut meats. On rice and nutmeat substrates, A. flavus was completely overgrown by members of the natural flora; even when A. flavus spores were present in superior numbers. Aflatoxin was not detected in the substrates of these cultures. / Graduation date: 1974

No description available.

No description available.

Graduation date: 1973

Thesis (Ph. D.)--Michigan State University. Dept. of Food Science and Human Nutrition, 2007. / Title from PDF t.p. (viewed on Apr. 16, 2009) Includes bibliographical references (p. 273-296). Also issued in print.

Thesis (M.S.)--University of Wisconsin--Madison, 1983. / Typescript. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.