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Citrinin and ochratoxin A disrupt renal function in many animal species. The mechanism(s) underlying these actions is (are) unclear. Although citrinin has been shown to bind covalently to renal tissue, there also is evidence that it is active in the unmetabolized form. Altered calcium homeostasis has been suggested as an event which might mediate cell injury and/or death; a possible role for calcium in citrinin- or ochratoxin A-induced nephrotoxicity is reported here. Renal cortical slice calcium balance was monitored by the uptake of 45Ca. Either ochratoxin A or citrinin added to fresh renal cortex slices enhanced 45Ca accumulation. These effects were evident as early as 5 min after addition of the toxins. Greater 45Ca uptake occurred with bathing solution calcium concentration of 1.1 mM than in the absence of added carrier calcium. Finally, the effect of citrinin to reduce p-aminohippurate accumulation by renal cortical slices was greater in the presence of calcium than in its absence.

Seven cases of Reye's syndrome in which aflatoxin B1 was isolated from the blood or liver or both are presented. In two cases aflatoxin B1 was found in the blood during the acute phase of the disease; a finding not previously reported. In six cases aflatoxin B1 was recovered from autopsy specimens of liver. A number of case reports linking aflatoxin B1 to Reye's syndrome have appeared in the literature but until now only one case had been reported from the United States. Aflatoxin B1 and its possible role in the etiology of Reye's syndrome is discussed. It is concluded that Reye's syndrome is the result of multiple interrelated factors.

Alterations in the hepatic microsomal monooxygenase system and in the concentrations of rubratoxin B in urine and feces were examined in male mice pretreated with corn oil, phenobarbital or 3-methylcholanthrene (3MC) and then given a single i.p. dose of rubratoxin B (1 mg/2.5 ml propylene glycol/kg). Twenty-four hours later the following parameters were determined; hepatic cytochrome P-450 content, enzyme activities of NADPH-cytochrome c reductase, NADPH-dependent dehydrogenase, aniline hydroxylase and ethylmorphine N-demethylase, and hepatic microsomal protein and reduced glutathione levels. Excretion of rubratoxin B in urine and feces also was determined. Rubratoxin B reduced the elevated cytochrome P-450 (136%, 134%) and protein (128%, 112%) to control values in animals pretreated with phenobarbital or 3MC, respectively; whereas, in the corn oil pretreated group, the mycotoxin reduced cytochrome P-450 by 38%. Aniline hydroxylase activity was reduced 31% or more in all pretreated animals. Rubratoxin B did not affect ethylmorphine N-demethylase activity in mice pretreated with phenobarbital; however, the enzyme activity was decreased significantly in the 3MC group. Rubratoxin B reduced the hepatic glutathione level in animals receiving 3MC (33%) or corn oil (22%). More rubratoxin B was detected in the urine than in the feces regardless of animals from the 3MC group. These data suggest a greater effect of rubratoxin B in the 3MC pretreated mice than in the phenobarbital pretreated animals.

The potential of Kathon biocide, an aqueous solution containing, as active ingredients (a.i.), a mixture of 5-chloro-2-methyl-4-isothiazolin-3-one and 2-methyl-4-isothiazolin-3-one (14.4% a.i.), to produce delayed contact dermatitis, a sensitization response, was evaluated in outbred Hartley guinea pigs by a modified Buehler's occluded epicutaneous patch technique. The relationship of the response as a function of induction/elicitation concentrations was investigated. Groups of guinea pigs received 9 induction doses of the biocide, 3 times a week, at concentrations ranging from 25-2000 ppm a.i. These guinea pigs were challenged with the biocide at concentrations ranging from 20-2000 ppm a.i., and the application sites were scored for erythema 24 and 48 h after the challenge. The incidence of delayed contact dermatitis in induced guinea pigs was dependent on both the induction and challenge concentrations. The EC50 (concentration at which delayed contact dermatitis was seen in 50% of the population) for induction at a challenge concentration of 2000 ppm a.i., a nonirritating concentration, was estimated to be 88 ppm a.i. with a slope of 3.47 probits/unit log concentration. The EC50 for elicitation at an induction concentration of 1000 ppm a.i. was estimated to be 429 ppm a.i. with a slope of 2.74 probits/unit log concentration. These data demonstrate that for Kathon biocide, there is an induction/elicitation concentration dependency for delayed contact dermatitis response, and there is a "no response concentration" zone where the biocide can be used without concern for clinically significant delayed contact dermatitis. In comparison with a previous study, these data also suggest that the number of induction doses may be an important factor in demonstrating the sensitization potential of a chemical.

Patulin (4-hydroxy-4H-furo[3,2-c]pyran-2(6H)-one), a carcinogenic lactone produced as a major metabolite by several fungi, inhibited the Mg++-dependent Na+-K+ activated adenosine triphosphatase (ATPase) activity of mouse brain microsomal fractions with an estimated IC50 of 3.0 X 10(-4) M. Inhibition was concentration dependent. Hydrolysis of ATP was linear with both time and enzyme concentration either with or without patulin in reaction mixtures. Altered pH and activity curves for Na+-K+ ATPase demonstrated comparable inhibition by patulin in buffered acidic ranges through an optimum of 7.5, followed by a reduction of toxicity to this system at higher alkaline pH. Kinetic studies of cationic-substrate activation of Na+-K+ ATPase indicated noncompetitive inhibition with respect to ATP (at low affinity nucleotide-directed sites) and Na+ (in the presence of low, noninterfering concentrations of K+). Competitive inhibition with respect to activation of the Na+-k+-stimulated activity and K+-stimulated p-nitrophenyl phosphatase activity of the enzyme system was indicated by altered binding site parameters without change in apparent Vmax in the presence of patulin. Activity was partially restored by washing. Preincubation of patulin with dithiothreitol or glutathione protected the enzyme from inhibition. Results suggest that patulin exerted its effect on Na+-K+ ATPase either directly by interfering with K+ binding or indirectly by inducing a conformational change in the enzyme.

Teratogenic effects of secalonic acid D (SAD), a toxic fungal metabolite, produced by P. oxalicum has been studied in rats. Crystalline SAD was injected as a single subcutaneous dose (25 mg/kg) on one of the gestation day 6-10, 12 or 14. Pregnant rats were treated with 15 mg/kg SAD on gestation day 10. Both doses produced teratogenic and fetotoxic effects, although the effects produced with the lower dose were less marked. Treatment on days 9 and 10 resulted in increased fetal resorptions and decreased fetal body weights. The highest number of resorptions, greatest depression of fetal body weights and largest number of malformations occurred when SAD was injected on day 10. Anophthalmia (days 9 and 10), exencephaly (day 9) and defects in limbs, digits and tail (day 10) were the major gross malformations. The main internal soft tissue defects were hydronephrosis (days 9 and 10) and tracheo-esophageal fistula and renal agenesis (day 10). Major skeletal defects involved the vertebrae and ribs. For all of the abnormalities observed, administration of SAD on day 10 of gestation produced the most marked effects in rats.

The metabolism of penicillic acid (PA), a carcinogenic mycotoxin, was studied in vitro using subcellular fractions of mouse-liver homogenates. PA reacted with glutathione (GSH) both enzymatically and non-enzymatically. Each reaction was of equal importance. The in vitro metabolism of PA using different hepatic subcellular fractions was essentially non-enzymatic when GSH was absent, but metabolism was strikingly increased when GSH was available. In the microsomal preparation in the presence of GSH 75% of the added PA was biotransformed within 30 min to metabolite(s) that were not extractable with organic solvents. HPLC analysis indicated that the metabolite(s) were more polar than the parent compound.

The LD50 for rubratoxin B dissolved in dimethylsulphoxide and administered to Syrian golden hamsters by ip injection was 0.4 (0.2-0.8) mg/kg body weight. The greatest number of deaths occurred 6-24 hr after administration. Gross alterations consisted of congestion of the liver, spleen and kidneys and histopathological alterations involved congestion of the spleen and congestion and mild degenerative changes in hepatocytes. In a second study, rubratoxin B was administered ip daily for 1 wk at doses of 25, 50 and 75% of the ip LD50. Mortality was greatest in the 50 and 75% dose groups. Toxicity was cumulative with multiple doses. Gross alterations were similar to those found in the LD50 study. Histopathological alterations included renal tubular degeneration and necrosis and focal necrosis of hepatocytes. The morphopathogenesis of lesions following a single ip LD50 dose was evaluated in a third study. Histopathological alterations were limited to the kidney and were characterized by renal tubular degeneration and necrosis. Renal lesions were first seen at 2 hr after administration and increased in severity to a maximum at 20 hr. Tubular regeneration was first seen at 24 hr and was found to the end of the test period (72 hr). Serum activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) and serum concentrations of total and indirect bilirubin were increased by 8 hr after dosing and returned to control values by the end of the test period. In a fourth study, rubratoxin B was administered ip daily for 1 wk at a dose of 25% of the ip LD50. Gross alterations were similar to those in the other studies. Histopathological alterations included progressive renal tubular degeneration and necrosis. Serum activities of AST and ALT and concentration of blood urea nitrogen (BUN) were progressively increased with increasing numbers of doses. Urinalysis indicated progressive renal tubular damage.

Ochratoxin A has been suggested to be an important, environmentally prominent nephrotoxin. Possible involvement in both human and animal disease states has been indicated. This study was designed to examine under controlled laboratory conditions, the nephrotoxic potential of ochratoxin A in the rat. Daily administration of ochratoxin A in doses of 0.75 and 2.0 mg/kg caused persistent urinary hypoosmolality coupled with excessive glucose and protein excretion. In addition the renal slice transport of several organic compounds also was altered. Renal slice tissue electrolytes and water were unaffected by ochratoxin A pretreatment. Direct addition of ochratoxin A to fresh renal cortex slices also depressed transport of some organic compounds. Although rats pretreated with ochratoxin A lost large amounts of body weight, the weight loss alone did not account totally for the changes in renal function. These data indicate that ochratoxin A has the capability of producing alterations in renal function suggestive of nephrotoxicity. However, the pattern of response is different than that observed with citrinin.

An ideal topical decontaminant for chemical warfare agents should be highly efficacious, easy to use, and have low toxicity. For toxicity assessment, a stepwise approach linking type of toxicity study to stage of decontaminant development is both efficient and economical. In the initial exploratory phase, range-finding acute toxicity studies using a few animals will suffice to screen out highly toxic decontaminants . More definitive acute toxicity studies and a short term mutagenic test are sufficient during the live agent efficacy testing phase. When an efficacious decontaminant is found and is ready to be formulated into a product, a more extensive toxicologic program including a 2-week dermal toxicity study, a skin sensitization study, and an inhalation study (for aerosol or powder formulations) should be implemented before clinical trials.

Renal function and transport have been examined in male and female rats at various times during a 6-month exposure to sodium saccharin. The animals were maintained on special diets containing 0%, 1%, 5% or 7.5% sodium saccharin. Developmental patterns for renal transport and renal function in the control animals were normal. The 5% and 7.5% concentrations of saccharin caused a reduction in the renal slice steady-stable accumulation of p-aminohippurate (PAH) and tetraethylammonium (TEA) which became significant after the animals were 30 days of age or older. Enhanced sodium excretion was observed in every age group of animals tested when these animals consumed sodium saccharin concentrations greater than or equal to 1% in the diet. At 60 days of age, increased urine volume, a decrease in urine osmolality, and increased potassium excretion were also found. All effects of saccharin on renal function and transport were promptly and completely reversible when the animals were removed from the saccharin diet for as little as 24 h. The effects of saccharin on PAH transport apparently were attributable to competitive inhibition. Although effects on TEA transport and other renal function parameters were not defined as to mechanism, the effects were as readily reversible as those on PAH when saccharin was removed from the diet.

Studies were undertaken to determine if the toxicity and pathology of rubratoxin B, a nephrotoxic and hepatotoxic mycotoxin, could be altered by increasing or decreasing the tissue concentrations of nonprotein sulfhydryls in the liver and kidneys. Rubratoxin B dissolved in DMSO was administered ip to weanling male Syrian hamsters and Mongolian gerbils at doses of 0.4 mg/kg and 2.0 mg/kg, respectively. Rubratoxin B caused a 70% decrease in hepatic and a 60% decrease in renal cortical nonprotein sulfhydryl (NPSH) concentration, an index of tissue reduced glutathione concentration, in both species. Treatment with cysteine prior to rubratoxin B administration did not greatly alter the decreases in NPSH concentration, but did decrease the severity of renal lesions. Treatment with diethyl maleate prior to rubratoxin B administration caused an 80% reduction in hepatic and a 70% reduction in renal NPSH concentration, which was prolonged by rubratoxin B treatment. The incidence and severity of renal lesions was increased in rubratoxin B-treated hamsters and gerbils given diethyl maleate compared to animals given rubratoxin B alone. Additionally, hepatic lesions were seen in hamsters and were more frequent and severe in gerbils that were treated with diethyl maleate prior to rubratoxin B dosing compared to animals given rubratoxin B alone. Renal and hepatocellular NPSH concentration appears to decrease during rubratoxin B toxicosis in the hamster and gerbil, and appears to be contributory in lesion development, since lesions in the liver and kidneys were more severe in animals in which NPSH concentrations were reduced by treatment with diethyl maleate.

Toxicity of secalonic acid D was examined by using lethality, growth retardation, and histopathology as indexes. The ip LD50 values of 37, 31, and 27 mg/kg were obtained for Charles River CD-1, Texas (ICR), and Sprague-Dawley (CF-1) strains of mice, respectively. The ip LD50 was 52 mg/kg in female CD-1 mice. The iv LD50 was 25 mg/kg in CD-1 male mice. Oral LD50 values of 400 mg/kg in male CD-1 mice and 25 and greater than 400 mg/kg in Sprague-Dawley day-old and weanling (21 d) rats of both sexes, respectively, were obtained. Doses of 20 mg/kg or more ip retarded growth and doses of 30 mg/kg or more ip were lethal to CD-1 mice. Oral doses required to produce such effects in day-old rats were 5 and 20 mg/kg (or higher), respectively. All ip doses of secalonic acid D caused pulmonary atelectases and foccal peritonitis in male CD-1 mice. The latter involved surfaces of abdominal viscera and produced limited subcapsular necrosis of hepatic parenchyma. Exposure to a single lethal dose iv (25 mg/kg or more) of secalonic acid D caused limited hepatic portal necrosis but no peritonitis or other associated local effects observed in CD-1 male mice after ip exposure. Cytoplasmic liposis and loss of glycogen and RNA from hepatocytes were observed in a single mouse receiving 50 mg/kg iv. Death resulting from cardiac and/or pulmonary insufficiency was suggested by atelectasis, pulmonary hemorrhages and edema, and massive atrial dilation in mice that died after lethal ip or iv doses of secalonic acid D. Five daily sublethal ip doses in CD-1 male mice resulted in dose-dependent mortality (LD50, 11.5 mg/kg) indicating cumulative effects.

The mutagenic effects of fiteen mycotoxins on Salmonella typhimurium strains TA1535, TA1537 and TA1538 and Saccharomyces cerevisiae strain D-3 were tested. Only aflatoxin B1 and sterigmatocystin were mutagenic. Both were active against S. typhimurium strain TA1538 and S. cerevisiae strain D-3; however, both required activation by the hepatic S-9 enzyme preparation. A positive correlation between the other mycotoxins reported to be carcinogenic and the two in vitro test systems employed was not demonstrated in our hands.

The effects of the aflatoxins on ATPase activities in mouse and rat tissues were investigated in vitro. The hepatic oligomycin-sensitive (O.S.) Mg++ ATPase was inhibited significantly. The order of inhibition was G1 greater than B1 greater than G2 greater than B2. Mouse O.S. Mg++ ATPase was more sensitive than the corresponding rat enzyme. The oligomycin-insensitive (O.I.) Mg++ ATPase activities in rat and mouse liver were not altered. Although aflatoxins G1 and B1 were more potent inhibitors of hepatic O.S. Mg++ ATPase, no concentration-response was observed, whereas aflatoxins G2 and B2 inhibited enzyme activity in a concentration-dependent manner. Spectral analysis of aflatoxin G1 solutions suggested that solubility was not related to the observed effects. In addition, the effects of aflatoxin B1 and G1 on mouse brain microsomal Na+-K+ ATPase were examined. Although aflatoxin B1 was more potent that G1, both mycotoxins significantly inhibited enzyme activity in a concentration-dependent fashion.

Rubratoxin B, a metabolite of Penicillium rubrum, is a potent mycotoxin that produces hepatic mid-zonal necrosis. Using propylene glycol as the solvent, the acute LD50 (i.p.) in male mice was estimated at 1.42 mg/kg. Pretreatment with s.c. pellets of pentobarbital reduced this toxicity approximately 42 percent, while castration and SKF 525A pretreatment enhanced toxicity approximately 20 percent and 60 percent, respectively. These studies also demonstrated that rubratoxin B potentiated pentobarbital-induced narcosis and enhanced pentobarbital hypothermia. The effect of SKF-525A and pentobarbital on the LD50 value of rubratoxin B suggests that the toxicity of this mycotoxin in mice does not arise from a toxic metabolite but from the parent compound.

Penicillic acid (PA), a mycotoxin, is hepatotoxic. A study was undertaken to investigate its effects on hepatobiliary excretory function, using the anionic compounds indocyanine green (ICG), in mice and rats. Pretreatment with a single dose of PA (90 mg/kg, ip, an LD50 dose in both species) resulted in depression of ICG excretion in both species. This depression was dose- and time-dependent. Decreases of 42 and 57% in biliary excretion of ICG were observed in rats and mice 48 and 72 h after PA pretreatment, respectively. Although bile flow was depressed significantly when expressed in terms of body weight, it was not altered in mice when expressed in terms of liver weight. Bile flow was not affected in rats. While the serum ICG concentration was increased after PA treatment in both species, the liver ICG concentration was not affected. The liver-to-serum, bile-to-serum, and bile-to-liver ICG concentration ratios decreased in PA-treated animals. These data suggest that the PA-induced hepatobiliary excretory dysfunction may result from depression of both uptake of ICG into the liver and bile canlicular transport of ICG.

T-2 toxin administered ip to mice at 1.0 and 1.5 mg/kg caused significant maternal mortality and decreased prenatal survival. Fetal weights were reduced on days 7, 10, and 11. Gross malformations involved the tail and limbs, exencephaly, open eye and retarded jaw development particularly in fetuses from females treated on days 9, 10, and 11. Ribs and vertebral malformations were seen in fetuses exposed on days 9 and 10.

Rubratoxin B, an alpha, beta unsaturated lactone containing bisanhydride metabolite of certain toxigenic strains of penicillium molds, significantly inhibited in vitro brain microsomal Na+ - K+ adenosine triphosphatase from swine, mouse, and rat with IC50's of 6.76, 6.67, and 6.80 x 10(-6) M, respectively. Mitochondrial Mg++ oligomycin-sensitive ATPase from mouse liver was also inhibited with an IC50 of 6.30 x 10(-6) M rubratoxin B. Structural modification of the polyfunctional parent compound (rubratoxin B) to rubratoxin A (a gamma lactol replaces one of the anhydride moieties) or formation of the dihydro analogs of rubratoxins A and B (2,3 saturated delta lactone) decreased inhibition to all ATPase systems. The structure-activity relationship relative to ATPase inhibition was rubratoxin B greater than dihydrorubratoxin B greater than rubratoxin A greater than dihydrorubratoxin A. The order of reactivity by these analogs to brain microsomal Na+ -K+ ATPase from 3 mammalian species and to mouse hepatic mitochondrial Mg++ ATPase was similar, indicating no significant species variations. These results suggest that the previously demonstrated in vivo inhibition of adenosine triphosphatase preparations by rubratoxin B was the result of the intact parent compound, because chemical alteration of any one of the functional moieties (either the maleic anhydride ring or conjugated lactone) resulted in significantly decreased inhibition of ATPase activity.

The LD50 for rubratoxin B dissolved in dimethylsulphoxide and administered to ICR mice by ip injection was 0.31 (0.22-0.43) mg/kg body weight. Gross alterations consisted of congestion of the liver and spleen and pallor and mottling of the kidneys. The histopathological alterations seen were hepatic and splenic congestion and renal tubular degeneration. The morphopathogenesis of lesions following a single ip LD50 dose was evaluated in a second study. Hepatic lesions were observed in mice killed between 8 and 40 hr after dosing and included diffuse sinusoidal congestion with mild sinusoidal ectasia, leucostasis, multifocal cytoplasmic vacuolation and necrosis of individual hepatocytes. Renal lesions were mild, not time-dependent, and consisted of mild degenerative changes in tubular epithelial cells of the outer stripe of the outer zone of the medulla. The activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in serum were increased 2 hr after dosing, peaked at 4 hr and returned to control values by the end of the test period. In a third study, rubratoxin B was administered ip daily for 1 wk at doses of 25, 50 and 75% of the ip LD50. Toxicity was dose related and cumulative with multiple doses at the highest dose. In a fourth study, rubratoxin B was administered ip at a dose of 75% of the ip LD50 daily for 1 wk. Histopathological alterations included hepatic congestion and mild sinusoidal ectasia, multifocal necrosis of hepatocytes, splenic congestion and mild renal tubular degeneration. Serum activities of AST and ALT were increased after multiple doses of rubratoxin B.

Kathon biocide (KB), a 75:25 mixture of 5-chloro-2-methyl-4-isothiazolin-3-one (KI) and 2-methyl-4-isothiazolin-3-one (KII), is a broad-spectrum antimicrobial agent. The absorption and disposition of 14C label were studied in male Sprague-Dawley rats following iv or dermal administration of KB 14C labelled in the carbonyl carbon of either KI or KII. KI-labelled Kathon was distributed rapidly following an iv dose (0.8 mg/kg). Total 14C label in the plasma was rapidly eliminated, with the data best described by a three-compartment model. Total 14C label concentration in the blood, however, remained constant at 3 ppm from 6 to 96 hr after administration and represented 29% of the dose, indicating that KI-labelled Kathon and/or metabolites was sequestered by the cellular fraction of blood. Elimination of 14C label from the tissues examined was biphasic, with a terminal half-life of more than 4 days; by 96 hr, faeces, urine and CO2 accounted for 35, 31 and 4% of the dose, respectively. Following a single 24-hr dermal application of 0.2 ml of an aqueous solution containing 2000 ppm [14C]KB (400 micrograms), rats absorbed 94% of KI-labelled KB and 82% of KII-labelled KB. However, the systemic bioavailability of KB was substantially less than this, since approximately half of the absorbed KB was associated with the skin at the application site 24 hr after the application. Percutaneous absorption was not affected by concentration over the range 500-4000 ppm. As the concentration of KI-labelled KB in the applied solution increased from 500 to 4000 ppm, the relative amount of 14C label associated with the skin decreased, while that in the excreta increased, indicating greater systemic penetration at higher concentrations. Low amounts of 14C label were found in the testes (less than 2 ppb) and blood (24 ppb) 28 days after a single dermal application of KI-labelled KB. Four consecutive daily applications of KI-labelled KB did not influence the proportion of the dose absorbed from the skin. However, the proportion of the dose excreted was higher than after a single application of an equivalent amount of KB.

Secalonic acid D (SAD) is an acutely toxic, teratogenic and possibly mutagenic fungal metabolite produced in corn by Penicillium oxalicum. Using ultraviolet absorbance at 340 nm as a means of detection, SAD was resolved as a sharp peak by reversed-phase high-performance liquid chromatography (HPLC) on a small particle (10 mu m) mu Bondapak C18 column in 4 min by an acetonitrile--water--glacial acetic acid-tetrahydrofuran (5:3:0.5:0.5 for solvent system A and 4:3:0.5:0.5 for solvent system B) elution solvent system. The flow-rates for the two solvent systems (A and B) were 1.5 and 1.7 ml/min; column operating pressures of 1500 and 1800 p.s.i., respectively, resulted. The relationship between peak height or peak area and the quantity of SAD injected was linear over a range of 1--50 ng. Detection was very sensitive with lower limits of detection of 0.6 and 0.7 ng of SAD in solvent systems A and B, respectively. Retention times, peak heights and peak areas were highly reproducible in both solvent systems. Detection of SAD in spiked (0.2--10 mu g/ml) urine and bile samples injected without cleanup onto the HPLC column and quantitative extraction of SAD by ethyl acetate from spiked, acidified plasma samples (0.1--5 mu g/ml) followed by HPLC analysis were obtained. Solvent system B gave better resolution of SAD from the interfering substances in bile and urine than solvent system A. However, sensitivity was slightly greater in solvent system A.

The disaggregation of liver polyribosomes (disruption of the site of protein synthesis) into single ribosomal units has been observed after treatment of mice with rubratoxin B, a toxic metabolite of Penicillium rubrum. Several mechanisms for the polysomal alteration were evaluated. A generalized decrease in mouse liver RNA following administration of rubratoxin B was noted. RNAase activity in the postmicrosomal fraction was increased after similar treatment; however, this increase was too small to account for the observed polysomal breakdown. Incubation of rubratoxin B with isolated polysome fractions indicated no direct interaction between the toxin and the polyribosomal complex, apparently eliminating such a complex as the cause of the in vivo disaggregation. Rubratoxin caused shifts in the ultraviolet absorption spectra of DNA and RNA in vitro, a preliminary indication of binding of the toxin to nucleic acid. Such preliminary observations lend support to the hypothesis that rubratoxin-induced polyribosomal disaggregation may result from an alteration in the critical informational macromolecules such as nucleic acids, or protein, or both in that both are involved in the control of cell growth. This alteration in the DNA-protein complex may then result in a malfunction of the DNA-dependent RNA polymerase activity and subsequent polysomal disaggregation.

The LD50 for rubratoxin B dissolved in dimethylsulphoxide and administered to Mongolian gerbils by ip injection was 2.0 (2.26-1.77) mg/kg body weight. The gross alterations observed at autopsy were pallor and mottling of the kidneys and liver and congestion of the spleen. The histopathological alterations seen were renal tubular degeneration and necrosis, degenerative changes in hepatocytes, and congestion of the spleen. The morphopathogenesis of lesions after a single ip LD50 dose was evaluated in a second study. The histopathological alterations that were observed were focal degeneration and necrosis of hepatocytes and renal tubular degeneration and necrosis. Hepatic lesions were observed in gerbils killed between 2 and 12 hr after dosing and included multifocal cytoplasmic vacuolation and coagulative necrosis of hepatocytes. The renal lesions were first observed 2 hr after dosing and increased to maximum severity at 40 hr after dosing. Tubular regeneration accompanied ongoing tubular necrosis at the end of the test period. The activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in serum were increased 4 hr after dosing, peaked at 24 hr and remained elevated to the end of the test period. Serum K+ concentration was increased 16 hr after dosing and remained elevated to the end of the test period. In a third study, rubratoxin B was administered ip once daily for 7 days at doses of 25, 50 or 75% of the ip LD50. Toxicity was dose related and cumulative with multiple doses. Histopathological alterations included renal tubular degeneration and necrosis, mild tubular dilation and focal necrosis of hepatocytes. In a fourth study, rubratoxin B was administered ip at a dose of 25% of the ip LD50 once daily for 7 days. Histopathological alterations included renal tubular degeneration, mild renal tubular dilation and focal necrosis of hepatocytes. Activities of AST and ALT in serum were slightly increased after multiple doses of rubratoxin B. Results of urinalysis indicated hepatic and renal tubular damage.

The in vivo effects of patulin on hepatic monooxygenase enzymes in male mice were examined after single and multiple exposures (i.p.) to the mycotoxin. The animals were exposed to a single dose of 1, 3 or 4.5 mg patulin/kg or daily doses of 1.0 mg/kg for 5 or 14 days. After single exposure no significant effect was observed on hexobarbital hydroxylation while aniline hydroxylase increased significantly at 3 mg/kg and at 48 hr. The demethylation of aminopyrine and ethylmorphine increased significantly 48 and 72 hr after 3.0 or 4.5 mg patulin/kg doses. A 12% increase was observed in cytochrome P-450 content 48 hr after exposure to 1 or 3 mg/kg. NADPH-cytochrome C reductase was enhanced significantly at all 3 dose levels at 48 hr. At 24 hr hepatic NADPH-dependent dehydrogenase activity increased 38 and 46% in animals exposed to a single dose of 3 mg patulin/kg, respectively. No effect of patulin was observed on the hepatic drug metabolizing enzymes examined except hexobarbital oxidation in mice exposed to multiple doses of patulin. Patulin at best was a weak inducer of the hepatic mixed function oxidase system.

With the exception of occasional reports of skin irritation, 20 years of commercial nitrofen use has not produced indications of toxicity in man. In mature non-pregnant laboratory animals nitrofen is only slightly toxic after acute oral, dermal, or respiratory exposures, and it is not a sensitizer. However, absorption through skin occurs rapidly from solvent-based formulations. Chronic administration in the diet at doses of 20 mg/kg body wt/day and higher produced liver toxicity in mice, rats, and dogs with liver tumors developing in mice at dose levels at 470 mg/kg/day. In addition to liver tumors in mice, the National Cancer Institute's Carcinogen Bioassay Program also found a dose-related incidence of pancreatic tumors in females of 1 of 2 strains of rat after lifetime feeding at levels at and above 65 mg/kg/day. Single and repeated doses given during pregnancy to rats and mice produce neonatal lethality accompanied by signs of impaired breathing, diaphragmatic hernias, heart anomalies, hydronephrosis, and apparent eye anomalies which are due to effects on the Harderian gland. These anomalies were produced by both oral and dermal doses, but did not occur in the rabbit or when dosing was restricted to the male parent only. Neonatal deaths appear after repeated maternal doses of 3 mg/kg/day and higher; the overall no observed effect level for effects in the offspring was 0.17 mg/kg/day. Based on a 10(-6) level of tumorigenic risk the acceptable average daily intake for man is 1 microgram/kg/day; pregnant women should not be exposed to more than 1.7 micrograms/kg in any single 24-h period.

Teratogenic effects of a single subcutaneous dose of ochratoxin A (1.75 mg/kg) on one of gestation days 4-10 has been studied in rats. Treatment on days 4, 5, 6 or 7 resulted in increased fetal resorptions, and decreased fetal weights. Fetal weights also were depressed after treatment on day 8. The highest number of resorptions, greatest depression of fetal weights and largest number of malformations occurred when ochratoxin A was injected on days 5, 6 or 7. External hydrocephaly, omphalocele, and anophthalmia were the major gross anomalies. Internal hydrocephaly and shift in position of esophagus were the main internal soft tissue defects. Major skeletal defects involved sternebrae, vertebrae and ribs. Subcutaneous injection of 1.75 mg/kg ochratoxin A seemed to be the minimum effective, single teratogenic dose. Lower doses (0.5 or 1.0 mg/kg) failed to produce any embryotoxicity or malformations and higher doses (e.g. 5.0 mg/kg) caused all fetuses to be resorbed.

Citrinin is a toxic metabolite produced by several species of Penicillium and Aspergillus. Citrinin is nephrotoxic and has been implicated in disease outbreaks in animals and humans. Citrinin was resolved in a sharp peak by reversed-phase high-performance liquid chromatography on a small-article (10 micrometers) column by elution in 4.25 min with a phosphoric acid (0.25 N)-acetonitrile-2-propanol solvent (55:35:10). Detection was by ultraviolet absorbance at 340 nm. The relationship between peak height and area and quantity injected was linear over a range of 2--50 ng at 340 nm and 5--200 ng at 365 nm. Retention time and peak area were highly reproducible. As little as 2--5 ng citrinin was detectable. Complete recovery of citrinin from plasma samples containing known quantities of [14C]citrinin was obtained over a range of 5--40 micrograms/ml by treatment of the plasma with 1 N hydrochloric acid followed by extraction with ethyl acetate. The method provides for the direct analysis of citrinin in urine and bile without prior extraction.

Piglets and calves were dosed orally with pure diacetoxyscirpenol (DAS) and T-2 toxin, crude extracts of Fusarium tricinctum containing T-2 toxin, and whole cultures of F tricinctum containing T-2 toxin at a constant daily rate of 0.1 mg toxin per kg body-weight (piglets) or 0.2 mg toxin per kg body-weight (calves). The treatment continued for periods of seven to 78 days but it failed to induce clinical haemorrhagic syndromes. Increasing the dose of F tricinctum culture five-fold for eight days following 78 days at the lower dose was equally ineffective. The lack of an effect by daily intakes of toxin that could have been ingested with naturally contaminated feedstuffs suggests that DAS, T-2 toxin and other metabolites of F tricinctum probably have little or no part to play in the aetiology of feed associated haemorrhagic disease.

The effect of ip administrated aflatoxin B1 and rubratoxin B, singly and in combination, on dogs was determined by serum tests, by observations of clinical signs and survival times, and by evaluation of gross and microscopic lesions. The dog is sensitive to the toxic effects of both mycotoxins. Glutamic-oxaloacetic transaminase, lactic dehydrogenase and alkaline phosphatase activities and survival time varied in relation to dose and to the mycotoxin(s) administered. All three plasma enzymes were elevated regardless of dose with the combination of aflatoxin B1/rubratoxin B at 24 hr after dosing, except LDH, which was within the normal range but only at the lowest dose level. Several serum constituents including BUN, cholesterol, uric acid, and total bilirubin were elevated, whereas serum glucose was depressed in dogs treated with the multiple-toxin regimen; these changes were not seen in dogs given only aflatoxin B1 but were characteristic in rubratoxin-treated animals. In general, gross findings at necropsy were similar in all dogs regardless of the dose regimen. A striking similarity existed in the histologic changes observed between lesions experimentally induced by the mycotoxin combination and those lesions reported for dogs fed toxic feed in laboratory studies or in natural cases of hepatitis X. Of particular similarity were the severe kidney lesions observed in dogs exposed to the mycotoxin combination and kidney lesions reported in natural outbreaks of hepatitis X. There can be little doubt of an association between hepatitis X and aflatoxin B1, although it is apparent that the disease probably involves more than a single toxic factor. Our results suggest that hepatitis X in dogs includes aflatoxin B1 as a primary etiological factor but that rubratoxin B also may be involved.

The effect of intraperitoneally administered penicillic acid, a mycotoxin produced by several species of Penicillium and Aspergillus, on female dogs of mixed breeding was determined by serum tests, by observation of clinical signs and survival times, and by evaluation of gross and microscopic lesions. Combination studies employing penicillic acid and a second mycotoxin, rubratoxin B, also were undertaken. Post mortem examination disclosed hemorrhaging of the serosal surfaces of the abdomen of dogs receiving penicillic acid. The most significant histologic change observed in penicillic-acid-treated dogs was congestion and dilatation of hepatic sinusoids. Extensive hepatic changes of the liver were noted only in the dog receiving 20 mg/kg penicillic acid. There was no evidence of parenchymal necrosis in any of the liver samples examined from animals given penicillic acid. A predominently peripheral lobular depletion of glycogen in parenchymal cytoplasm also was seen in liver sections from animals exposed to penicillic acid. Although slight decreases in lactic dehydrogenase were observed, no trends were detected in the several blood enzymes and serum constituents examined that could be specifically related to penicillic acid intoxification. Glutamic-oxaloacetic transaminase, lactic dehydrogenase and alkaline phosphatase activities and survival time varied in relation to duration of exposure and total dose of rubratoxin B administered. The lesions in animals injected with 1.0 mg/kg rubratoxin B consisted of mild hepatic necrosis and degenerative changes in renal tubular epithelium. An additive effect due to the combined administration of penicillic acid and rubratoxin B was observed only by an elevation in serum sodium and chlorine levels.

Kathon biocide, an aqueous solution containing a mixture of 5-chloro-2-methyl-4-isothiazolin-3-one and 2-methyl-4-isothiazolin-3-one in an approximate ratio of 3:1, was tested for mutagenic activity in Salmonella typhimurium, L5178Y mouse lymphoma cells in culture and Drosophila melanogaster. Tests also were conducted for chromosome aberrations in vivo on mouse bone marrow cells, for DNA damage/repair in primary rat hepatocytes in culture, and for morphological transformation in C3H 10T1/2 cells in culture. Kathon biocide produced point mutations in the absence of a rat-liver metabolizing system in bacteria (strain TA 100) and mammalian cells in culture. In the presence of rat-liver metabolizing system a 10-fold higher concentration was required to induce point mutations in mammalian cells in culture. No mutagenic activity was observed with the metabolizing system and S. typhimurium. Negative results were obtained in the sex-linked recessive lethal assay in Drosophila, the in vivo cytogenetic assay in mice, the unscheduled DNA synthesis assay in cultured rat hepatocytes, and the in vitro cell transformation assay.