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A rapid method for the determination of aflatoxins was developed using high pressure liquid chromatography and a radial compression separation system. A standard solution of aflatoxins B1, B2, G1, G2, and M1 was analyzed solution of aflatoxins B1, B2, G1, G2, and M1 was analyzed at flow rates of 2.0 and 6.0 mL/min. Retention times, peak heights, and peak areas were reproducible over a 3-day period. Coefficients of variation for aflatoxin B1 at 2.0 and 6.0 mL/min were, respectively, 1.04 and 0.87% (retention time); 2.9 and 4.7% (peak height); and 8.2 and 4.7% (peak area). At 6.0 mL/min there was an approximate 25% loss in sensitivity but a greater than 50% reduction in retention time. Separation of all the aflatoxins was excellent using a dual flow rate of 2.0 mL/min with a change to 8.0 mL/min at 15 min post-injection. The applicability of the radial compression separation system for the rapid determination of aflatoxins in human tissues was also tested. Spiked samples of liver, serum, and urine-showed good resolution of all aflatoxin peaks at the higher flow rates.

The mycotoxin ochratoxin A (OA) was derivatized to an O-methyl,methyl ester (Me2) with diazomethane and then determined by high pressure liquid chromatography (HPLC). Both OA and OA-Me2 were chromatographed by reverse phase HPLC with a mobile phase of acetonitrile-water (60 + 40). An increase in retention time of 309 s was observed with OA-Me2 which was detectable at 254 nm at levels as low as 3 ng. Recovery of OA as OA-Me2 from chicken kidney homogenates and human plasma was quantitative following simple extraction and cleanup procedures, reaction with diazomethane, and HPLC analysis. The novel method described should prove useful for measuring and confirming OA in tissues and in further studies on the biological fate of this mycotoxin.

Technical grade nitrofen was fed to 4 groups (25/group) of male Sprague-Dawley rats for 13 weeks at dietary concentrations of 0, 100, 500, or 2500 ppm. Untreated female rats of the same age and strain were mated with treated males and fertility, gestation, and lactation indices were monitored. After mating, males were bled for clinical chemistry and hematology analyses, killed, and necropsied; organ weights were recorded, and liver, testes, and kidneys evaluated histopathologically. Offspring were observed for 35 days to determine general health and viability. At 2500 ppm, body weights of paternal animals were reduced throughout the dosing period. No hematologic abnormalities were seen. Statistically significant changes noted in clinical chemistry parameters included increased total protein, albumin, globulin, and cholesterol and decreased glucose. Testes, kidneys, and liver weights were increased at 500 and 2500 ppm, but histologic changes were limited to the liver with hypertrophy and cytoplasmic basophilia of centrilobular hepatocytes occurring at 500 and 2500 ppm. There were no effects on fertility, gestation, litter size, weight, or sex ratio in any group. Offspring health and survival to day 35 was unaffected. Based on the parameters examined, the no-observed effect level (NOEL) for male rats fed nitrofen for 13 weeks was 100 ppm. Male reproductive performance was not affected at dietary concentrations up to and including 2500 ppm.

Penicillic acid (PA) is a mycotoxin with reported cytotoxic, cardiotoxic, and carcinogenic activity and it can occur in high concentration in corn. The occurrence of PA in contaminated poultry feed represents a potential public health hazard. A reverse phase high pressure liquid chromatographic (HPLC) method is proposed for determining PA residues in chicken tissues. Optimization of chromatography was achieved for PA using a mobile phase consisting of acetonitrile: H2O. PA was detected by ultraviolet absorption at 254 nm, identified by retention time, and quantitated by peak area integration. Blood, parenchymal tissues, muscle, and alimentary tract contents were homogenized, sonicated, and acid treated followed by extraction with ethyl acetate and analysis by HPLC. Acute oral dosing of chickens with PA over a range of 50 to 550 mg/kg body weight resulted in detectable levels of the mycotoxin (confirmed by gas liquid chromatography) in gizzard muscle and contents, liver, kidney, heart, and intestinal contents. This method should prove useful both for the rapid and sensitive detection of PA residues in poultry and in further studies on the distribution and metabolism of this mycotoxin.

Teratogenicity and fetotoxicity of secalonic acid D, a toxic fungal metabolite produced by Penicillium oxalicum, were investigated with pregnant CD1 mice. The compound was administered ip on d 7-15 of pregnancy. A dose-dependent reduction in weight gain of mothers receiving all doses of secalonic acid D and an increase in resorptions of implanted embryos of dams treated with more than 5 mg/kg secalonic acid D occurred. The latter effect was nearly 100% at 15 or 9 mg/kg given in NaHCO3 with or without dimethyl sulfoxide (DMSO), respectively. A corresponding decrease in the percent of live fetuses and a decrease in the average fetal body weight on d 19 of pregnancy also occurred. Multiple gross, skeletal, and visceral anomalies were noted in fetuses born to mothers receiving 10 mg/kg or more in NaHCO3 containing DMSO. In NaHCO3 alone, the minimum teratogenic dose was 6 mg/kg. Major malformations included cleft palate, cleft lip, open eyelids, missing phalangeal ossification centers, and shortened mandibles. The results indicated that secalonic acid D is embryocidal and teratogenic as well as fetotoxic when given to female CD1 mice during pregnancy.

Ten smokers participated in a study to compare the absorption of nicotine from the smoke aerosol of a new cigarette that heats, but does not burn tobacco (test) with a cigarette that burns tobacco (reference). The average plasma nicotine concentrations obtained by the 7th test cigarette (13 ng/ml) and 7th reference cigarette (24 ng/ml) were proportional to the nicotine yielded by the two cigarettes as determined under Federal Trade Commission machine-smoking conditions. These data demonstrate that the smoke aerosol obtained by smoking a cigarette which heats tobacco produces plasma profiles of nicotine that are similar to the profiles obtained from smoking a cigarette that burns tobacco.

Substances that cause liver damage in humans were identified through a literature search conducted on Toxline and Medline. Using the same search strategy, species other than man were selected, in whom hepatic injury could be attributed to exposure to the identified substances. A total of 38 substances were identified as producing liver damage, manifested by either clinical chemistry or histopathology. The substances included 24 drugs, 9 industrial chemicals, 3 environmental agents, 1 pesticide, and ethanol. Twelve of the 36 compounds have been toxicologically evaluated in man, rodent, and non-rodent. Histopathologic liver damage was reported in all three species for 11 of these compounds. Only carbencillin produced histopathologic damage in man but not in either the rodent or non-rodent. Where clinical chemistry changes were reported for all three species categories, only eight substances induced similar reactions in all three species. Only with two substances did man and rodent react similarly (both positive), while the non-rodent was negative. The two substances were polychlorinated biphenyl (PCB) and tetrachlorethane. In the majority of the cases in which either or both histopathologic or clinical chemistry changes were reported for man or for the rodent or non-rodent, the changes that occurred were qualitatively similar. The rodent was as sensitive a predictor for hepatic effects as the non-rodent. Although it is impossible to predict how liver damage observed in a laboratory animal is correlated with human liver damage, hepatotoxicity in the rodent must be considered as indicative of potential hepatic damage in man.

Eight groups of 30 male and 30 female rats were exposed 1 hr per day, 5 days per week for 13 weeks, to smoke from reference (tobacco burned) or test (tobacco only heated) cigarettes, at nicotine concentrations of 5, 15, or 30 micrograms/liter of air. Similar smoke concentrations of wet total particulate matter and carbon monoxide were produced in each of the test/reference comparisons. There was a pronounced depression of minute ventilation of animals in the reference groups, but not in the test animals. Blood carboxyhemoglobin concentrations were similar in animals exposed to smoke from test and reference cigarettes. Plasma concentrations of nicotine and cotinine in the test groups were higher than in the reference groups. There were no differences between the smoke-exposed groups in terms of body weight or feed consumption. At necropsy, an increase in heart weight was noted in both high exposure groups. There were notable differences in histopathology, with fewer and less-pronounced changes in the test groups than in the reference groups. Many of the histopathological responses induced in the reference groups were absent in the test groups. Overall, the study demonstrated a substantial reduction in the biological activity of smoke from the test cigarette when compared with the reference.

Rubratoxin B was given to Syrian hamsters as a single intraperitoneal dose of 0.4 mg/kg. Hamsters were killed at 1, 2, 4, and 6 hr after dosing, and the kidneys and liver were fixed by intravascular perfusion. Renal ultrastructural alterations were evident at 1 hr after treatment and were limited to the straight portion of the proximal tubule. The most prominent alterations were brush border disruption, dilation of smooth endoplasmic reticulum, mitochondrial swelling, cytoplasmic rarefaction, myelin figure formation, and swelling of basal interdigitating processes. Renal alterations were suggestive of damage to membrane structure and/or transport functions and interference with cellular bioenergetics. Hepatic alterations were not seen in the rubratoxin B-treated hamsters of this study.

Twenty isolates of Penicillium oxalicum produced secalonic acid as their major secondary metabolite. Fermentation conditions were determined for toxin production in grain and liquid media. The 50% lethal dose value for mice ranged from 26.5 to 51.7 mg/kg dependent on animal strain and sex, males being more susceptible than females. Secalonic acid was nontoxic and nonteratogenic to the chicken embryo and exhibited poor antibiotic properties. Its potential role in mycotoxicoses is discussed.

Teratogenicity of daily intraperitoneal (i.p.) injections of 1.5 or 2.0 mg/kg of patulin on days 6 through 17 of pregnancy and mutagenic effects of acute i.p. exposure of 3.0 mg/kg of patulin on male germ cells were evaluated. Resorption of all implanted embryos occurred at 2.0 mg/kg/day of patulin, while a significant reduction in the average body weight of 19-day-old fetuses from patulin-treated mothers, compared to control fetuses, was noticed at 1.5 mg/kg/day without any lethal effects on the implanted embryos. Patulin was embryocidal, possibly fetotoxic but was neither teratogenic nor mutagenic to mice.

In an episode of aflatoxicosis in feeder pigs, mortality was about 20%. Histopathologic findings characteristic of experimentally induced aflatoxicosis and the finding of aflatoxin B1 in the serum of pigs and in the cornbased feed confirmed the diagnosis. Aflatoxins B1 and B2 were found in the corn used to prepare the feed. Combine harvesting of the corn, which cracked a large percentage of the kernels, coupled with prolonged drying time of the corn probably contributed to the aflatoxin production. Although the corn was fed to adult swine without observable effect, 47 of the 250 feeder pigs developed typical signs of aflatoxicosis. Unseasonably cold weather apparently was a factor in initiating the onset of clinical signs and probably increased the severity of the disease.

The pharmacokinetics of penicillic acid (PA), a carcinogenic mycotoxin, was investigated in male mice. Absorption of PA after po administration of [14C]PA was rapid. Only a small percentage of the radioactivity in the plasma was unchanged PA. After ip or iv administration of [14C]PA (90 mg/kg), blood, liver, kidneys, intestine, lungs, heart, and spleen contained the largest amounts of radioactivity while brain tissue accumulated the least. Over 90% and approximately 60% of the administered radioactivity was excreted in the urine after iv and ip injection, respectively, but essentially no unchanged PA was detected in the urine. Over 25% of the administered radioactivity following an iv dose of [14C]PA (90 mg/kg) was excreted in the bile in 60 min; no unchanged PA was detected in the bile. The excretion of radioactivity in the bile was decreased in diethyl maleate-pretreated mice. Only a small amount of the administered radioactivity was recovered in the feces and as expired CO2. The unchanged PA concentration-time curve in plasma was best fit by three, two, and one compartment open models after iv, ip, and po administration, respectively. Based on these results, it was concluded that metabolism and not excretion of unchanged parent penicillic acid is the major process of elimination of PA from the blood. There are extensive route-dependent differences in the kinetic behavior of PA.

The fungal toxin, citrinin, has been implicated as a nephrotoxin. Both in vitro and in vivo studies were undertaken to examine, in a controlled laboratory setting, the effects of citrinin on overall renal function as well as on specific renal transport processes. At a dose of 70 mg/kg citrinin caused an increase in urine production and a decrease in urine osmolality in the Sprague-Dawley rat. Maximal effects were observed at 4 days after a single dose, and animals surviving for that time period regained normal renal function by 5--8 days. Animals that failed to survive first entered a period of oliguria or anuria. Fischer 344 rats did not demonstrate an unusual sensitivity to citrinin. Renal slice transport of various organic compounds, e.g., paminohippurate (PAH) and tetraethylammonium (TEA), also was inhibited with maximal effects seen at day 4. In vitro effects of citrinin on fresh renal tissue were different, in part, from the effects seen with pretreatment. No alterations of renal cortical inorganic electrolytes were observed.

Citrinin has an inhibitory effect on growth of T. pyriformis. Citrinin also caused a shift to smaller cell size, particularly at the higher concentrations (25, 50 and 100 mug/ml). The mycotoxin exerted only a marginal effect on the respiration of T. pyriformis; but citrinin (25 mug/ml) induced an inhibitory effect on DNA, RNA and protein content. The greatest decrease was in RNA, while smaller decreases in protein and DNA were observed. A bioassay employing T. pyriformis was determined; the lower limit of citrinin detection was between 1 and 5 mug/ml.

Pregnant golden hamsters were injected ip with 2.5-20 mg/kg ochratoxin A on one of gestation days 7-10. The largest dosages when given on day 7,8, or 9 increased prenatal mortality and on day 9 diminished fetal growth. Malformations, such as micrognathia, hydrocephalus, short tail, oligodactyly, syndactyly, cleft lip, micromelia, and heart defects occurred, but no skeletal malformations were noted. These results add to the prenatal effects of ochratoxin A previously reported in mouse, rat, and chicken embryos.

The distribution and excretion of radioactivity from [14C]citrinin (3 mg/kg, i.v) was determined in male rats. At 0.5 h after administration maximum values of 14.7% and 5.6% of total radioactivity were observed in the liver and kidneys, respectively, and by 6 h decreased to 7.5% in the liver and 4.7% in the kidney. Plasma concentration of 14C decreased from 9.2% at 0.5 h to 4.7% at 6.0 h. 2 plasma elimination rates were observed, with half-lives of 2.6 and 14.9 h, respectively. Approximately 80% of the administered 14C activity was excreted in feces and urine by 24 h after administration. A second group of rats was pretreated with 50 mg/kg of citrinin, i.p., 4 days prior to administration of 3 mg/kg [14C]citrinin, i.v. 30% of the pretreated animals died and the remaining animals were divided into 2 groups on day 4 after pretreatment; rats which were "nephrotoxic" and rats which had "recovered" from the initial insult of citrinin. Proteinuria and glucosuria as well as enhanced urine output were observed in "nephrotoxic" rats 4 days after pretreatment. 24 h after [14C]citrinin, only 13% of 14C activity was detected in the urine of "nephrotoxic" rats. The plasma disappearance curve had 2 elimination rates, with half-lives of 0.6 and 14.1 h. "Nephrotoxic" rats retained 7.5% of the administered radioactivity in the liver compared to 1.3% in the "recovered" rats 24 h after the tracer dose and 47% of the radioactivity was either excreted in feces or in the colon contents after 72 h compared to 17.5% in "recovered" rats. Extraction of urine samples from "nephrotoxic" and "recovered" rats with chloroform suggested increased water soluble metabolites of citrinin in the urine from "nephrotoxic" rats. These data also suggested that in normal rats the kidneys are the major route of elimination of citrinin and its metabolite(s) while in rats rendered nephrotoxic by citrinin pretreatment, elimination is more dependent on hepatic excretion.

Toxicologists and pathologists are often faced with the dilemma of categorizing changes observed in the respiratory tract of laboratory animals as either "adaptive" or "toxic." However, it is often difficult to interpret the nature of a given change as either "adaptive" or "toxic." Certain lesions or changes in the respiratory tract are to be expected from the concentration of materials given or the experimental design of a study. Careful analysis suggests that some of these changes may be more properly described as adaptive rather than toxic within the context of a given study or situation. Tissue changes discussed in this paper include squamous metaplasia of laryngeal epithelium, goblet cell change in respiratory epithelium, macrophage accumulation within alveoli, and bronchiolization of alveolar epithelium. Examples provided show that some of these changes observed in inhalation studies are similar in severity but slightly increased in frequency over sham control animals. The introduction of exogenous material into the respiratory tract of laboratory animals in an experimental setting should be expected to result in certain changes. The challenge scientists must accept is to interpret these changes so that toxic events may be separated from adaptive changes. In order to meet this challenge, studies incorporating several species and novel technologies may have to be utilized.

Secalonic acid D is an acutely toxic and teratogenic mycotoxin, produced by Penicillium oxalicum in corn. Two rodent mutagenicity tests, the dominant lethal test designed to evaluate male germ cell mutations and the micronucleus test designed to evaluate somatic cell mutations, were conducted in mice to assess the carcinogenic potential of secalonic acid D. Data obtained indicate that the positive control compound triethylene melamine (TEM) induced dominant lethal mutations during weeks 1 through 4 after administration to male mice and also resulted in a greatly increased population of micronucleated polychromatic (immature) erythrocytes in bone marrow of treated male mice. Secalonic acid D, however, failed to induce dominant lethal mutations but produced a slight but statistically significant increase in the number of micronucleated polychromatic erythrocytes in mouse bone marrow at maximally tolerated doses. It is concluded that secalonic acid D may e weakly mutagenic in a mouse somatic cell mutation system.

Recent work has shown that V accumulates in the kidney and is a potent inhibitor of Na+, K+-adenosinetriphosphatase (ATPase) in vitro. Thus, as a nutritionally required element, V may regulate cation transport. The effect of chronic intake of the metal on Na+, K+-ATPase in vivo has not been reported. In this study laying strain chickens were fed calcium orthovanadate for 15 mo from d 1 of age at levels of 0, 25, 50, and 100 ppm in the diet. Whole tissue homogenates and 13,000 X g fractions were analyzed for ATPase activities. Concentrations of V producing 50% inhibition of Na+, K+-ATPase activity ranged from 1.0 X 10(-5) M in liver to 1.8 X 10(-6) M in kidney, which was the most sensitive tissue tested in vitro. Mg2+ -ATPase was more resistant to V than Na+, K+-ATPase. Studies in vivo suggested a V-dependent inhibition of renal Na+, K+-ATPase. Correlation of enzyme specific activity and levels of V in kidneys suggested V-ATPase mediated alteration in renal function.

The effects of cooking methods on the in vitro mutagenicity of individual foods, the in vitro mutagenicity of meals containing those foods, and the mutagenic exposure of human volunteers following consumption of the meals were examined using Ames bacterial strain TA98 with S-9 metabolic activation. Three methods of food preparation--boiling, baking and frying/flame-broiling--were compared. With meats, frying or broiling resulted in higher in vitro mutagenicity (10- to 50-fold) than did baking or boiling, whereas for carbohydrates, eggs or vegetables mutagenicity did not vary markedly with cooking method. The observed (experimental) mutagenic activity of the meals was quite similar to their calculated (predicted) mutagenicity, obtained by summing the mutagenicity of the individual foods in the meal. The close agreement between experimental and predicted mutagenicity indicated that components of the meal did not interact in either a synergistic or inhibitory manner. The mutagenicity of fried flame-broiled meals was approximately 10-fold greater than the mutagenicity of baked or broiled meals, which were similar in mutagenicity. The mutagenicity of human urine following consumption of the meals was related to the in vitro mutagenicity of the meals themselves. The in vitro mutagenicity of meals is markedly affected by the cooking method used to prepare them and the mutagenicity of the diet may be reflected in the mutagenicity of body fluids.

Penitrem A or an unknown tremorgenic toxin, "X," was produced by 10 of 60 fungal isolates obtained from a pasture involved in an outbreak in cattle and sheep resembling migram and ryegrass staggers. Tremorgenic properties of extracts containing penitrem A or toxin X were confirmed by bioassay.

The rubratoxins are toxic metabolites produced by Penicillium rubrum and P. purpurogenum on food and feedstuffs. Rubratoxin B is hepatotoxic, mutagenic and teratogenic. Rubratoxins A and B were resolved as sharp peaks in the order A-B by reversed-phase high-pressure liquid chromatography on a small-particle (10 micrometer) column in 3 min by an acetonitrile-water-ethyl acetate elution solvent (11:9.9:3), with detection by ultraviolet absorbance at 254 nm. The relationship between peak height and quantity injected was linear over a range of 0.25-5 microgram for rubratoxin A and 0.05-5 microgram for rubratoxin B. Retention time and peak height and peak area were highly reproducible for both toxins. Detection was very sensitive, allowing detection of 3-5 ng rubratoxin B, and 15-20 ng rubratoxin A. Quantitative recovery of rubratoxin B from spiked urine samples was obtained over a range of 5-40 microgram/ml, with maximum recovery from urine samples adjusted to pH 2 before extraction. Good recovery of rubratoxin B was also obtained from spiked plasma samples subjected to treatment with 3 N hydrochloric acid followed by extraction with ethyl acetate.

Secalonic acid D (SAD) is an acutely toxic and teratogenic fungal metabolite produced by Penicillium oxalium in corn and other cereal grains. Experiments were conducted to study the effects of single and multiple doses of SAD on pentobarbital-induced narcosis, as an index of the modulating effect of SAD on the hepatic drug metabolizing enzymes in mice. The effects of known enzyme modulators-phenobarbital (PB), 3-methyl cholanthrene (3-MC), and diethylaminoethyl diphenylproply acetate hydrochloride (SKF-525A)-on the acute toxicity of SAD in mice were also studied using body weights, mortality, and histopathology as indices. Results of this study failed to demonstrate any modulating effect by SAD of pentobarbital metabolizing enzymes. Pretreatment with SKF-525A, an enzyme inhibitor, enhanced SAD toxicity in mice, whereas pretreatment with PB or 3-MC, known enzyme inducers, had no effect. Further studies of interaction of [14C]SAD with PB and SKF-525A revealed that although neither PB nor SKF-525A altered the ratio of parent compound to total metabolites excreted in bile, SKF-525A significantly reduced the bile flow as well as the elimination of SAD-derived radioactivity in bile. These results strongly suggest the possibility that the effects of SKF-525A, other than the enzyme inhibition, may be responsible for its enhancement of SAD toxicity in mice.

Aflatoxin B 1 was detected, and its identity confirmed, in hypoallergenic milk substitutes composed, among other things, of the following ingredients susceptible to possible aflatoxin contamination: soya protein isolate, soy and coconut oils, cornstarch, and corn syrup. Except for one determination all findings were under 1 ng of aflatoxin/ml of formula at drinking concentration. Manufacturers' reserve samples of soya protein isolate, the ingredient thought to be the most likely source of the aflatoxin, were found to be uncontaminated. Reserve samples of other ingredients were not examined. Because hypoallergenic formula may be the major nutrient of an individual with a metabolic defect at infancy, a vulnerable stage of life, available control measures should be used to avoid aflatoxin-contaminated ingredients.

Methods were developed for bioproduction and extraction of rubratoxin B from liquid cultures of Penicillium rubrum P-13 (NRRL A-11785). A maximum of 874.7 mg of toxin per liter of medium was attained in 21 days using stationary cultures of Mosseray's simplified Raulin solution enriched with 2.5% malt extract. Malt extract was required for rubratoxin production. Rubratoxin was not produced in either shake flasks or in fermentors with restricted aeration. Crystalline toxin was obtained by liquid-liquid extraction of concentrated culture medium with ethyl ether. Adhering colored impurities were removed by column chromatography and by recrystallization from acetone.

Aspergillus flavus ATCC 15517 produced up to 212 mg per liter of total aflatoxin in submerged culture in aerated (3,000, 6,000, 9,000, and 12,000 ml/min) and agitated medium in 14-liter fermentors with 10 liters of medium consisting of 2% yeast extract and 10% sucrose. Aflatoxin production increased with time. A maximum of 212 mg/liter was produced at 9,000 ml/min aeration, whereas the yield decreased substantially at the lower aeration rates. Two other strains of A. flavus synthesized aflatoxin in smaller quantities.

Of the 133 microorganisms tested, Tetrahymena pyriformis and Volvox aureus were the most sensitive to rubratoxin B, being inhibited at 25 and 50 mug/ml, respectively.