Fusaric acid and modification of the subchronic toxicity to rats of fumonisins in F. moniliforme culture material.

Fumonisins and fusaric acid (FA) are mycotoxins produced by Fusarium moniliforme and other Fusarium which grow on corn. Fumonisins cause animal toxicities associated with F. moniliforme and, like F. monliforme, they are suspected human oesophageal carcinogens. Toxic synergism was obtained by simultaneous administration of FA and fumonisin B1 to chicks in ovo. To determine the effect of FA on in vivo toxicity of F. moniliforme culture material (CM), male rats (12 groups, n = 5/ group) were fed diets containing 0.025, 0.10 or 2.5% CM (providing dietary levels of 3.4, 18.4 or 437 ppm fumonisins, respectively) to which, at each CM level, 0, 20, 100 or 400 ppm FA were added. Additionally, an FA control group was fed 400 ppm FA only and an untreated control group was given neither FA nor culture material. Apoptosis and other effects consistent with those caused by fumonisins were present in the kidneys of animals fed 0.025% or more CM and in the livers of animals fed 2.5% CM. FA was without effect. No differences between the untreated and FA control groups were noted and no differences among the four groups (0-400 ppm FA) fed 0.025% CM, the four groups fed 0.10% CM or the four groups fed 2.5% CM were apparent. Thus, FA exerted no synergistic, additive or antagonistic effects on the subchronic in vivo toxicity of fumonisin-producing F. moniliforme.     

Biological and chemical characterization of metabolites of Fusarium moniliforme isolates

Metabolites of fusarium moniliforme isolates from different types of corn were characterized biologically and chemically. The biological assays included rat feeding, rat dermal tests and inoculation of embryonated eggs. Thin-layer chromatography, gas liquid chromatography, and mass spectrophotometry were used to isolate and determine their chemical identities. The metabolites were identified as diacetoxyscripenol, ipomeanol, ipomeanine and diplodiatoxin. The biological tests revealed significant weight loss in rats fed the contaminated corn for 5 w. Hemorrhages and edema in the brain and intestine were detected in all the groups of rats.     

Inhibition of protein synthesis in rat primary hepatocytes by extracts of Fusarium moniliforme-contaminated corn

Mycological screening of two separate lots of corn samples that caused field cases of equine leukoencephalomalacia (ELEM) revealed heavy contamination with the fungus Fusarium moniliforme. Neutral and acidic fractions of a chloroform-methanol (1:1, v/v) extract of the corn were evaluated for toxicity using rat primary hepatocytes. The extracts had little effect on the release of lactate dehydrogenase from the hepatocytes, and were without effect on unscheduled DNA synthesis, indicating low cell lethality and lack of genotoxicity. However, neutral extracts of the corn were found to contain potent inhibitor(s) of protein synthesis as measured by incorporation of [3H]valine into the hepatocytes. When an isolate of F. moniliforme obtained from the corn samples or an isolate of F. moniliforme from South Africa that had previously been shown to cause ELEM (MRC 826) were grown on autoclaved seed corn, neutral extracts of the culture materials similarly inhibited protein synthesis. Whether the compound(s) responsible for inhibition of protein synthesis is associated with any of the toxic syndromes associated with F. moniliforme remains to be determined. The use of primary hepatocytes may be a useful bioassay for elucidating biologically active secondary metabolites of fungi.     

Equine leucoencephalomalacia (ELEM): a study of Fusarium moniliforme as an etiologic agent

Signs and lesions characteristic of equine leucoencephalomalacia were produced in one of two donkeys given corn cultured with Fusarium moniliforme Sheldon. Gross and histopathologic lesions of the cerebrum included an extensive necrotic cavitation within one cerebral hemisphere, disruption and rarefication of the subcortical white matter, prominent perivascular hemorrhage, and some mononuclear cell perivascular cuffing. Another donkey and three rabbits fed the cultured corn did not develop characteristic signs or lesions of the toxicosis. Chick bioassay studies indicated that the cultured corn which produced the disease did not contain a monovalent salt of moniliformin.     

Toxicology and carcinogenesis studies of isophorone in F344 rats and B6C3F1 mice

Toxicology and carcinogenesis studies of isophorone were conducted by administering 0, 250, or 500 mg/kg body weight per day by gavage in corn oil to groups of 50 F344/N rats and 50 B6C3F1 mice of each sex, 5 days/week, for 103 weeks. Dosed male rats developed proliferative lesions of the kidney including hyperplasia, adenoma, and adenocarcinoma of the renal tubule, and epithelial hyperplasia of the renal pelvis. Non-proliferative kidney lesions observed in dosed male rats included mineralization, and a more severe nephropathy in low dose animals than in controls or high dose animals. Carcinomas of the preputial gland occurred in high dose male rats. No isophorone-related lesions were observed in female rats. In male mice, isophorone exposure may have been associated with an increase in hepatocellular neoplasms and mesenchymal neoplasms of the integumentum in high dose animals, and with a marginally increased incidence of lymphoma in low dose male mice. In mice, no non-neoplastic lesions in males or females, or neoplastic lesions in females were considered associated with isophorone administration.     

Urinary Physiologic and Chemical Metabolic Effects on the Urothelial Cytotoxicity and Potential DNA Adducts ofo-Phenylphenol in Male Rats

ortho-Phenylphenol (OPP), a fungicide and antibacterial agent with food residues, is carcinogenic to rat bladder. The present studies provide information on changes in urinary composition and urinary metabolites, urothelial cytotoxicity and regenerative hyperplasia, and DNA adducts in male F344 rats fed OPP. An initial experiment evaluated dietary doses of 0, 1,000, 4,000, and 12,500 ppm OPP fed for 13 weeks. There was no evidence of urinary calculi, microcrystalluria, or calcium phosphate-containing precipitate, but urothelial cytotoxicity and hyperplasia occurred at the highest dose only. In a second experiment, rats were fed dietary OPP levels of 0, 800, 4,000, 8,000, and 12,500 ppm. Urinary pH was >7 in all groups. Urinary volume was increased at the 2 highest doses with consequent decreases in osmolality, creatinine, and other solutes. Total urinary OPP metabolite excretions were increased, mostly excreted as conjugates of OPP and of phenylhydroquinone. Free OPP or free metabolites accounted for less than 2% excreted in the urine without a dose response. Urothelial toxicity and hyperplasia occurred only at doses of 8,000 and 12,500 ppm. OPP–DNA adducts were not detected in the urothelium at any dose. In summary, OPP produces cytotoxicity and proliferation of the urothelium at dietary doses ≥8,000 ppm without formation of urinary solids. The paucity of unconjugated metabolites and the lack of OPP–DNA adducts suggests that OPP is acting as a bladder carcinogen in male rats by inducing cytotoxicity and hyperplasia without it or its metabolites directly binding to DNA.     

Vehicle and mode of administration effects on the induction of aberrant crypt foci in the colons of male F344/N rats exposed to bromodichloromethane

Bromodichloromethane (BDCM) and tribromomethane given by corn oil gavage were previously found to induce neoplasia in the large intestine of rats. Our chronic bioassay of BDCM administered in drinking water failed to produce colon neoplasia in male F344/N rats. We recently reported that BDCM induces aberrant crypt foci (ACF), putative precursor lesions in the development of colon cancer, when included in the drinking water of male rats. To investigate whether ACF induced by BDCM could be promoted by corn oil (CO), male F344/N rats were exposed to 0.7 g BDCM/L in drinking water or 50 mg BDCM/kg body weight by oral gavage in CO. Animals exposed to drinking water, CO, or 15 mg/kg azoxymethane (AOM) (ip) constituted the negative, vehicle, and positive controls. After 26 wk, colons were examined for ACF. A significant decrease in water consumption was observed in both the positive controls and BDCM-treated animals; however, no difference was noted in final body weight. The administration of CO to AOM-exposed animals produced a significant increase in total ACF when compared to AOM alone. BDCM produced a significant increase in ACF when compared to control, but no difference was noticed between BDCM exposure by oral CO gavage and control. Additionally, no difference was noted between BDCM exposure by drinking water and by oral CO gavage. This study demonstrates that the formation of ACF is independent of the route of BDCM exposure (drinking water vs. oral corn oil gavage), with both routes producing similar ACF values of 1.33 +/- 0.49 and 1.5 +/- 0.51 ACF/colon.     

Toxicology and carcinogenesis studies of fumonisin B1 (cas no. 116355-83-0) in F344/N rats and B6C3F1 mice (feed studies)

[formula: see text] Fumonisin B1 is a mycotoxin produced by the fungus Fusarium moniliforme, one of the major species found in corn. There are no known commercial or medical uses of fumonisin B1. Fumonisin B1 was nominated by the FDA Center for Food Safety and Applied Nutrition for study because of its occurrence in corn and corn-based products in the United States and its toxicity in field exposure of horses and pigs. Male and female F344/N Nctr BR rats and B6C3F1/Nctr BR (C57BL/6N x C3H/HeN MTV-) mice were exposed to fumonisin B1 (92% pure) in feed for 28 days or (greater than 96% pure) for 2 years. 28-DAY STUDY IN RATS: Groups of 10 male and 10 female rats were fed diets containing 0, 99, 163, 234, or 484 ppm fumonisin B1 for 28 days. There were no exposure-related deaths in rats. The mean body weights of the 484 ppm groups were significantly less (-16%) than those of the controls. Dietary concentrations of 99, 163, 234, and 484 ppm fumonisin B1 resulted in average daily doses of 12, 20, 28, and 56 mg fumonisin B1/kg body weight for males and females. Additional groups of male and female rats were exposed to the same concentrations of fumonisin B1 for 28 days for clinical pathology studies. The concentrations of creatinine, cholesterol, triglycerides, and total bile acids, as well as activities of the enzymes alanine aminotransferase, alkaline phosphatase, aspartate aminotransferase, and gamma-glutamyltransferase, were generally significantly greater in the 484 ppm groups than in the control groups at all time points, indicating hyperlipidemia and a hepatic effect. Fumonisin B1 is an inhibitor of ceramide synthase, resulting in an interruption of de novo sphingolipid synthesis. This enzyme inhibition results in increased levels of sphinganine (or increased sphinganine:sphingosine ratio) in tissues and urine. Urinary sphinganine was increased in groups of males exposed to 163 ppm or greater, while urinary sphinganine was increased in all exposed groups of females. The kidney weights, relative to body weight, of all exposed groups of rats were less than those of the control groups, decreasing by approximately 11% in the females and 20% in the males. Apoptosis and degeneration of the kidney were observed in all exposed males and in most females exposed to 163 ppm or greater. The incidences of minimal to mild apoptosis, degeneration, and mitotic alteration of the liver were significantly increased in 234 and 484 ppm males and in females exposed to 163 ppm or greater. The incidences of bile duct hyperplasia were significantly increased in males and females in the 484 ppm groups. In the core study, male rats in all exposed groups and females exposed to 163 ppm or greater had significantly increased percentages of hepatocytes in one or more proliferative (non-G0) states. 28-DAY STUDY IN MICE: Groups of 12 male and 12 female mice were fed diets containing 0, 99, 163, 234, or 484 ppm fumonisin B1 for 28 days. There were no exposure-related deaths in mice. The mean body weights of the 484 ppm groups of males were significantly less than those of the controls. Feed consumption by males exposed to 484 ppm was less than that by the controls; dietary concentrations of 99, 163, 234, and 484 ppm fumonisin B1 resulted in average daily doses of approximately 19, 31, 44, and 93 mg/kg for males and 24, 41, 62, and 105 mg/kg for females. Additional groups of male and female mice were exposed to the same concentrations of fumonisin B1 for 28 days for clinical pathology studies. Cholesterol and total bile acid concentrations and alanine aminotransferase and alkaline phosphatase activities were increased at 484 ppm, indicating hyperlipidemia and a hepatic effect. Urinary sphinganine concentrations and sphinganine/sphingosine ratios were increased in 484 ppm male mice. In 484 ppm males and all exposed groups of females, the incidences of hepatocellular necrosis, diffuse periportal hypertrophy, and diffuse centrilobular hyperplasia, as well as hyperplasia of the bile canaliculi and Kupffer cells, were generally significantly greater than those in the controls. Core study males exposed to 99, 163, or 234 ppm had significantly increased incidences of hepatocellular cytoplasmic alteration. Hepatocytes of 484 ppm male mice and all exposed groups of female mice were induced into proliferative (non-G0) states. 2-YEAR STUDY IN RATS: Groups of 48 male and 48 female rats (40 for 5 ppm groups) were fed diets containing 0, 5, 15, 50, or 150 ppm fumonisin B1 (males) or 0, 5, 15, 50, or 100 ppm fumonisin B1 (females) (equivalent to average daily doses of approximately 0.25, 0.76, 2.5, or 7.5 mg/kg to males and 0.31, 0.91, 3.0, or 6.1 mg/kg to females) for 105 weeks. Additional groups of four male and four female rats were exposed to the same concentrations as the core study animals and were evaluated at 6, 10, 14 or 26 weeks. Survival, Body Weights, and Feed Consumption Survival, mean body weights, and feed consumption of exposed male and female rats were generally similar to the controls throughout the study. Clinical Pathology Findings Sphinganine/sphingosine ratios were increased in the urine of 15, 50 and 150 ppm males and 50 and 100 ppm females exposed to fumonisin B1 for up to 26 weeks. The sphinganine/sphingosine ratios were also increased in kidney tissue of 50 and 150 ppm males (85- and 119-fold) and 50 and 100 ppm females (7.8- and 22-fold) at 2 years. Cell Proliferation Analyses Renal tubule epithelial cell proliferation was increased in 50 and 150 ppm male rats exposed to fumonisin B1 for up to 26 weeks. Renal tubule epithelial cell proliferation was marginally increased in 100 ppm females. Organ Weights and Pathology Findings Kidney weights of 50 and 150 ppm males were less than those of the controls at 6, 10, 14, and 26 weeks and at 2 years. Kidney weights of 100 ppm females were less than those of the controls at 26 weeks, and kidney weights of 15, 50, and 100 ppm females were less than those of the controls at 2 years. At 2 years, there was a significant increase in the incidences of renal tubule adenoma from none in the groups receiving 15 ppm or less to five of 48 in 150 ppm males. Renal tubule carcinomas were not present in male rats receiving 15 ppm or less and occurred in seven of 48 and 10 of 48 male rats in the 50 and 150 ppm groups, respectively. Incidences of apoptosis of the renal tubule epithelium were generally significantly increased in males exposed to 15 ppm or greater for up to 26 weeks. The incidences of focal renal tubule epithelial hyperplasia were significantly increased in 50 and 150 ppm males at 2 years. 2-YEAR STUDY IN MICE: Groups of 48 male and 48 female mice were fed diets containing 0, 5, 15, 80, or 150 ppm (males) or 0, 5, 15, 50, or 80 ppm (females) fumonisin B1 (equivalent to average daily doses of approximately 0.6, 1.7, 9.7, or 17.1 mg/kg to males or 0.7, 2.1, 7.1, or 12.4 mg/kg to females) for 105 weeks. Additional groups of four male and four female mice were exposed to the same concentrations as the core study animals and were evaluated at 3, 7, 9, or 24 weeks. Survival, Body Weights, and Feed Consumption Survival of males and females in the 15 ppm groups and of 5 ppm females was significantly greater and survival of 80 ppm males and females was significantly less than that of the control groups. Mean body weights and feed consumption of exposed mice were generally similar to the controls. Organ Weights and Pathology Findings Liver weights, relative to body weight, were increased 1.3- and 2.9-fold in 50 and 80 ppm females at 2 years. At 2 years, the incidences of hepatocellular adenoma in 50 and 80 ppm females were significantly greater than those in the controls and occurred with a positive trend. Similarly, the incidences of hepatocellular carcinoma increased from none in the groups receiving 0, 5, or 15 ppm fumonisin B1 to 10 of 47 females at 50 ppm and nine of 45 females at 80 ppm. The incidences of hepatocellular hypertrophy were significantly increased in 15, 80, and 150 ppm males and in 50 and 80 ppm females at 2 years. The incidences of hepatocellular apoptosis were significantly increased in 50 and 80 ppm females at 2 years. CONCLUSIONS: Under the conditions of these 2-year feed studies, there was clear evidence of carcinogenic activity of fumonisin B1 in male F344/N rats based on the increased incidences of renal tubule neoplasms. There was no evidence of carcinogenic activity of fumonisin B1 in female F344/N rats exposed to 5, 15, 50, or 100 ppm. There was no evidence of carcinogenic activity of fumonisin B1 in male B6C3F1 mice exposed to 5, 15, 80, or 150 ppm. There was clear evidence of carcinogenic activity of fumonisin B1 in female B6C3F1 mice based on the increased incidences of hepatocellular neoplasms. The sphinganine/sphingosine ratios were increased in the urine and the kidney tissue of rats receiving diets containing fumonisin B1. There was evidence of apoptosis and increased cell proliferation of the renal tubule epithelium in exposed rats, particularly in those groups of males that developed renal tubule neoplasms. Increased incidences of hyperplasia of the renal tubule epithelium also occurred in these groups of male rats. In mice exposed to the higher concentrations of fumonisin B1, males and females had increased incidences of hepatocellular hypertrophy and females had increased incidences of hepatocellular apoptosis.     

Comparison of the Hepatic and Renal Effects of 1,4-Dichlorobenzene in the Rat and Mouse

The effects of 1,4-dichlorobenzene (DCB) have been comparedin male F344 rats given 0 (corn oil control), 25, 75, 150, and300 mg/kg DCB and male B6C3F₁ mice given 0 (corn oil control),300, and 600 mg/kg DCB by daily oral gavage five days per weekfor 1, 4, and 13 weeks. The two highest rat and both mouse doselevels were the same as those employed In a NTP bioassay, whereDCB produced kidney tumors in male rats and liver tumors inmice. DCB produced significant dose-related increases in relativeliver weight in both the rat and the mouse which was associatedwith, respectively, mild and marked centrilobular hypertrophy.Administration of DCB also produced a sustained induction ofmicrosomal cytochrome P450 content and 7-pentoxyresorufin O-depentylaseactivity in both species. Western immunoblotting studies demonstratedthat DCB induced CYP2B isoenzyme(s) in both rat and mouse livermicrosomes. Replicative DNA synthesis was studied by implantingosmotic pumps containing 5-bromo-2'-deoxyuridine in study Weeks0–1, 3–4, and 12–13. In the rat hepatocytelabeling index values were only increased in animals given 300mg/kg DCB for 1 week, whereas hepatocyte labeling index valueswere significantly increased in mice given 300 and 600 mg/kgDCB for 1 and 4 weeks. DCB treatment produced significant increasesin rat renal P₁/P₂ proximal tubule cell labeling index valuesat all time points, whereas little effect was observed in mousekidney. The observed species difference in DCB-induced livertumor formation may reflect the greater sensitivity of the mouseto tumor promotion by a CYP2B inducer. For the kidney, the presentdata provides further evidence that while DCB-induced a nephropathyis associated with a sustained stimulation of cell replicationin male rat renal proximal tubule cells, this effect is notobserved in the male mouse.     

NTP toxicology and carcinogensis Studies of 2,4-hexadienal (89% trans,trans isomer, CAS No. 142-83-6; 11% cis,trans isomer) (Gavage Studies)

2,4-Hexadienal, a colorless to yellow liquid with a pungent "green" or citrus odor, is used as a food additive for flavor enhancement, as a fragrance agent, as a starting material or intermediate in synthetic reactions in the chemical and pharmaceutical industries, as a fumigant, and as a corrosion inhibitor for steel. 2,4-Hexadienal was nominated for study by the National Cancer Institute because of the potential for carcinogenicity based on its alpha,beta-unsaturated aldehyde structure and the potential link between exposure to lipid peroxidation products in the diet and human malignancies. The commercial product is a mixture containing chiefly trans,trans-2,4-hexadienal in equilibrium with cis,trans-2,4-hexadienal. Male and female F344/N rats and B6C3F1 mice received 2,4-hexadienal (89% trans,trans; 11% cis,trans) in corn oil by gavage for 16 days, 14 weeks, or 2 years. Tissues and plasma from dosed rats were examined for malondialdehyde and glutathione concentrations, and DNA adducts were characterized in liver and forestomach samples from dosed rats and mice. Genetic toxicology studies were conducted in Salmonella typhimurium, rat and mouse bone marrow cells, and mouse peripheral blood erythrocytes. 16-DAY STUDY IN RATS: Groups of five male and five female rats were administered 0, 3, 9, 27, 80, or 240 mg 2,4-hexadienal/kg body weight in corn oil by gavage, 5 days per week, for 16 days. Three male and three female 240 mg/kg rats died before the end of the study. Mean body weight gains of 240 mg/kg rats were significantly less than those of the vehicle controls. Clinical findings included diarrhea, ataxia, lethargy, and nasal/eye discharge in males, and lethargy, paleness, and abnormal breathing in females in the 240 mg/kg groups. Liver weights of 240 mg/kg females were significantly greater than those of the vehicle controls. Gross and microscopic lesions indicative of forestomach necrosis and ulceration were present in most 240 mg/kg rats, and forestomach epithelial hyperplasia was microscopically evident in most 80 mg/kg rats. 16-DAY STUDY IN MICE: Groups of five male and five female mice were administered 2,4-hexadienal in corn oil by gavage at doses of 0, 3, 9, 27, 80, or 240 mg/kg, 5 days per week, for 16 days. Chemical-related deaths occurred in one male and one female in the 240 mg/kg groups. Female mice in the 240 mg/kg group lost weight during the study. Gross and microscopic lesions indicative of forestomach necrosis and ulceration were present in all 240 mg/kg mice, and forestomach epithelial hyperplasia and hyperkeratosis were microscopically evident in 80 mg/kg mice. 14-WEEK STUDY IN RATS: Groups of 10 male and 10 female rats were administered 2,4-hexadienal in corn oil by gavage at doses of 0, 7.5, 15, 30, 60, or 120 mg/kg, 5 days per week, for 14 weeks. All rats survived to the end of the study. Mean body weights of 30, 60, and 120 mg/kg males were significantly less than those of the vehicle controls. The only clinical finding attributed to 2,4-hexadienal administration was hypersalivation in 30 and 120 mg/kg males and females. The incidences of forestomach hyperplasia and nasal olfactory atrophy or necrosis were significantly increased in 120 mg/kg rats. Nasal lesions occurred in most 120 mg/kg male rats. 14-WEEK STUDY IN MICE: Groups of 10 male and 10 female mice were administered 2,4-hexadienal in corn oil by gavage at doses of 0, 7.5, 15, 30, 60, or 120 mg/kg, 5 days per week, for 14 weeks. No deaths were attributed to administration of 2,4-hexadienal. Mean body weights of males and females were similar to those of the vehicle controls throughout the study. Clinical findings included salivation and anal wetness in males and females. Kidney weights of 60 and 120 mg/kg males and liver weights of 60 mg/kg males and females were significantly greater than those of the vehicle controls. The incidences of forestomach hyperplasia and/or nasal olfactory atrophy or necrosis were significantly increased in 120 mg/kg mice. 2-YEAR STUDY IN RATS: Groups of 50 male and 50 female rats were administered 2,4-hexministered 2,4-hexadienal in corn oil by gavage at doses of 0, 22.5, 45, or 90 mg/kg, 5 days per week, for up to 105 weeks. Survival of all dosed groups of rats was similar to that of the vehicle control groups. The mean body weights of 90 mg/kg males were generally less than those of the vehicle controls throughout the study. The incidences of squamous cell papilloma of the forestomach occurred with positive trends in male and female rats. This neoplasm was found in 58% of males and 34% of females in the 90 mg/kg groups. In the forestomach of male rats, papilloma multiplicity was increased in the 90 mg/kg group, and squamous cell carcinomas were found in one 45 mg/kg male and two 90 mg/kg males. Epithelial hyperplasia of the forestomach occurred in most 45 and 90 mg/kg rats. 2-YEAR STUDY IN MICE: Groups of 50 male and 50 female mice were administered 2,4-hexadienal in corn oil by gavage at doses of 0, 30, 60, or 120 mg/kg, 5 days per week, for up to 105 weeks. Survival of dosed mice was similar to that of the vehicle controls. The mean body weights of all dosed groups were generally similar to those of the vehicle controls throughout the study. The incidences of squamous cell papilloma of the forestomach occurred with positive trends in male and female mice; squamous cell carcinomas were present in 120 mg/kg males and females. Epithelial hyperplasia of the forestomach occurred in many 120 mg/kg mice. Two 120 mg/kg males had uncommon squamous cell carcinoma of the oral cavity (tongue). GENETIC TOXICOLOGY: 2,4-Hexadienal was mutagenic in S. typhimurium strain TA100 with and without induced hamster or rat liver enzymes; no mutagenic activity was detected with strains TA1535 or TA98, with or without S9. Results of bone marrow tests in male rats and male mice given intraperitoneal injections of 2,4-hexadienal showed a small increase in the induction of micronucleated erythrocytes. However, neither test was repeated, and the test results were judged to be inconclusive. Results of peripheral blood micronucleus tests in male and female mice treated with 2,4-hexadienal by gavage for 14 weeks were negative. CONCLUSIONS: Under the conditions of these 2-year gavage studies, there was clear evidence of carcinogenic activity* of 2,4-hexadienal in male and female F344/N rats and male and female B6C3F1 mice based on increased incidences of squamous cell neoplasms of the forestomach. The occurrence of squamous cell carcinoma of the oral cavity (tongue) in male B6C3F1 mice may have been related to the administration of 2,4-hexadienal. Hyperplasia of the forestomach in male and female rats and mice was associated with administration of 2,4-hexadienal. Synonyms: Hexa-2,4-dienal; 2,4-hexadienal; 2,4-hexadien-1-al; 2,4-Hx; 1,3-pentadiene-1-carboxaldehyde; 2-propylene acrolein; sorbaldehyde; sorbic aldehyde     

Contamination is a frequent confounding factor in toxicology studies with anthraquinone and related compounds

Anthraquinone (AQ) (9,10-anthracenedione) is an important compound in commerce. Many structurally related AQ derivatives are medicinal natural plant products. Examples include 1-hydroxyanthraquinone (1-OH-AQ) and 2-hydroxyanthraquinone (2-OH-AQ), which are also metabolites of AQ. Some commercial AQ is produced by the oxidation of anthracene (AQ-OX). In the recent past, the anthracene used was distilled from coal tar and different lots of derived AQ often contained polycyclic aromatic hydrocarbon contaminants, particularly 9-nitroanthracene (9-NA). Many toxicology studies on AQ used contaminated anthracene-derived AQ-OX, including a National Toxicology Program (NTP) 2-year cancer bioassay that reported a weak to modest increase in tumors in the kidney and bladder of male and female F344/N rats and in the livers of male and female B6C3F1 mice. The AQ-OX used in that bioassay was mutagenic and contained 9-NA and other contaminants. In contrast, purified AQ is not genotoxic. The purpose of this paper is to provide additional information to help iterpret the NTP cancer bioassay. This paper describes a quantitative analytical study of the NTP anthracene-derived AQ-OX test material, and presents the results of mutagenicity studies with the 1-OH-AQ and 2-OH-AQ metabolites and the primary contaminant 9-NA. Purified 1-OH-AQ and 2-OH-AQ exhibited only weak mutagenic activity in selected strains of tester bacteria and required S9. Literature reports of potent mutagenic activity for 1-OH-AQ and 2-OH-AQ in bacteria minus S9 are, once again, very likely the result of the presence of contaminants in the test samples. Weak activity and limited production of the 1-OH-AQ and 2-OH-AQ metabolites are possible reasons that AQ fails to exhibit activity in numerous genotoxicity assays. 9-NA was mutagenic in tester strains TA98 and TA100 minus S9. This pattern of activity is consistent with that seen with the contaminated AQ-OX used in the NTP bioassay. Analysis of all the mutagenicity and analytical data, however, indicates that the mutagenic contamination in the NTP bioassay probably resides with compounds in addition to 9-NA. 9-NA exhibited potent mutagenic activity in the L5178Y mammalian cell mutagenicity assay in the presence of S9. The positive response was primarily associated with an increase in small colony mutants suggesting a predominance of a clastogenic mechanism. Quantitative mutagenicity and carcinogenicity potency estimates indicate that it is plausible that the contaminants alone in the NTP AQ-OX bioassay could have been responsible for all of the observed carcinogenic activity. Although AQ-OX is no longer commercially used in the United States, many of the reported genotoxicity and carcinogenicity results in the literature for AQ and AQ derivative compounds must be viewed with caution.     

Metabolism of fusarin C by rat liver microsomes. Role of esterase and cytochrome P-450 enzymes with respect to the mutagenicity of fusarin C in Salmonella typhimurium

Fusarin C (FC) is a potent mutagen present on Fusarium moniliforme contaminated corn. This compound requires metabolic activation for which microsomes from phenobarbital-induced rats are most effective. Inhibition of the simultaneously induced esterase activity, which produced a less mutagenic metabolite, doubled the mutagenicity of FC. Carbon monoxide inhibited the mutagenicity of FC, suggesting the involvement of a heme containing enzyme. However, monoclonal antibodies specific for the phenobarbital-induced cytochrome P-450 enzymes PB-4 and PB-5, while inhibiting O-demethylation of p-nitroanisole and aflatoxin B1 mutagenicity, had not effect on FC mutagenicity. This implies that either these enzymes are not involved in the activation of FC or FC competes well with the antibodies for binding to the cytochrome P-450 enzymes. Two additional metabolites of FC were detected. One had an ultraviolet spectrum similar to FC: the other had a lambda max at 326 nm, and its retention time on reverse phase HPLC was very sensitive to changes in pH.     

Assessment of the estrogenic effects of zearalenone after treatment with ozone utilizing the mouse uterine weight bioassay

The ability of ozone gas (O3) to detoxify zearalenone (ZEN), a commonly occurring estrogenic mycotoxin, was assessed utilizing the mouse uterine weight bioassay. Solutions containing 12 ppm ZEN in water were ozonated for varying time periods (0, 0.5, and 5 min), then extracted with chloroform and evaporated to dryness. The residue was redissolved in acetonitrile and analyzed for ZEN. High-performance liquid chromatography (HPLC) analysis of aliquots indicated a rapid degradation and decline in parent ZEN level with increasing time of ozone treatment. The acetonitrile solution containing the degraded ZEN residue was added to a known volume of corn oil and evaporated under nitrogen to eliminate the acetonitrile in the oil. Eighteen-day-old prepubertal female mice (B6C3F1 strain) were gavaged daily with the test chemicals in 50 microl of corn oil between d 18 and 23. Initial dose-response studies showed that a concentration of 60 microg ZEN/mouse/d produced uterine weights that were significantly higher than the uterine weights of control animals (2.7 times higher than that of the solvent control). Treatment groups for the ozonation study included: DES, 0.1 microg (positive control), untreated ZEN (60 microg), extraction control for ZEN (60 microg), 0.5 min ozone-treated ZEN (60 microg), 5 min ozone-treated ZEN (60 microg), solvent control (50 microl), and absolute control. Results showed the uterine weights of animals receiving the ozone-treated ZEN were not significantly affected. These findings were in agreement with HPLC analyses and suggested that ozone can prevent the estrogenic effects of this important mycotoxin in mice. Importantly, ozone treatment of contaminated whole grains may enable the practical detoxification and control of ZEN. Also, the mouse uterine weight bioassay may be useful in assessing the efficacy of other detoxification strategies for estrogenic chemicals.     

Lifetime toxicity/carcinogenicity study of FD & C Red No. 3 (erythrosine) in rats

FD & C Red No. 3 was fed to Charles River CD rats as a dietary admixture in two long-term toxicity/carcinogenicity studies. The studies consisted of an in utero and an F1 phase. In the former, the compound was administered to five groups of the F0 generation rats (60 of each sex/group) at levels of 0.0, 0.0, 0.1, 0.5 or 1.0% ('original study') and 0.0 or 4.0% ('high-dose study'). The concurrent control groups received the basal diet. After random selection of the F1 animals, the long-term phase was initiated using the same dietary levels and 70 rats of each sex/group, including the three control groups. Rats were exposed for a maximum of 30 months. No compound-related effects were noted in the in utero phase. Mean body weights of the female F1 rats on 4.0% FD & C Red No. 3 (3029 mg/kg/body weight/day) were significantly lower than those of controls (P less than 0.01) throughout the study. Food consumption increased in all treated groups in a dose-related manner. There were no significant effects on the haematology, serum chemistry and urinalysis and no compound-related effects on survival. In male rats receiving 4.0% FD & C Red No. 3 (2464 mg/kg/day) thyroid weights were increased, with a mean weight of 92 mg compared to 44 mg for controls, and statistically significant increases in the incidence of thyroid follicular cell hypertrophy, hyperplasia and adenomas were recorded. A numerically increased incidence of thyroid follicular adenomas in female rats given 0.5, 1.0 or 4.0% FD & C Red No. 3 was not statistically significant. The no-observed-adverse-effect levels established in these studies were 0.5% (251 mg/kg/day) for male rats and 1.0% (641 mg/kg/day) for females.     

Effects of dietary fat level and aflatoxin B1 treatment on rat hepatic lipid composition

Sprague-Dawley rats were given either ten daily doses of aflatoxin B₁ (AFB₁) or the solvent tricaprylin intragastrically over a 2-wk period and were fed diets containing either 1.6 or 20% corn oil throughout the study. Hepatic lipid composition was analysed in groups of five rats both 3 and 13 wk after the start of treatment, in order to determine short-term and longer-term alterations. Total lipid and cholesterols (total, free and esterified) increased on the high-fat diet at wk 3. At wk 13 only total and esterified cholesterol were increased by 20% corn oil. AFB₁ treatment resulted in large intra-group variations in total lipid and cholesterol at wk 3, but these were no longer apparent by wk 13. AFB₁ produced various alterations in the fatty acid composition of hepatic phosphatidylcholine (PC) and phosphatidylethanolamine (PE), apparent at wk 3 but not at wk 13. The unsaturation index decreased but no changes were seen in the saturated fatty acids. Only in animals fed 20% corn oil did AFB₁ result in significant changes in 18:2, 20:3 and 22:6 fatty acids, while 20:4 and 22:5 tended to decrease and 18:1 to increase in response to AFB₁ treatment with both diets in both phospholipids. The high-corn oil diet was found to increase 18:2, 22:6, and total unsaturation in PC and PE, while the ratio of 20:4 to 18:2 tended to decrease in these phospholipids, γ-Glutamyltranspeptidase, an indicator of liver damage, was significantly increased in AFB₁-treated animals, with the greatest increase over controls in those fed the high-fat diet.     

Toxicology and carcinogenesis studies of p,p'-dichlorodiphenyl sulfone in rats and mice.

p,p'-Dichlorodiphenyl sulfone (DDS) is used as a starting material in the production of polysulfones and polyethersufones, a family of thermoplastics. DDS was studied because of its high production volume and use. In toxicology studies, 10 Fischer 344 rats and 10 B6C3F1 mice/sex/group were fed diets containing 0, 30, 100, 300, 1,000 or 3,000 ppm DDS for 14 weeks. All animals survived until the end of the studies. Mean body weights of groups exposed to 300 ppm or greater were significantly decreased. Liver and kidney in rats and liver in mice were the major target organs of DDS toxicity. Dose-related increases in liver weights and incidences of centrilobular hepatocyte hypertrophy were observed in DDS-exposed groups. Nephropathy was seen in male and female rats only at and above 300 ppm. Neurotoxicity evaluations were negative in DDS-treated animals. Clinical chemistry and hematology parameters were minimally affected. In the 2-year toxicity and carcinogenicity studies, 50 rats and 50 mice/sex/group were fed diets containing 0, 10 (male rats), 30, 100, or 300 ppm DDS for 104 to 105 weeks. Survival of exposed groups was not affected. There were no clinical signs of toxicity related to DDS exposure. Final mean body weights were 2-17% lower in DDS-treated groups. Liver was the only target organ of DDS-induced toxicity. The incidence of centrilobular hepatocyte hypertrophy in mice and rats, and the incidence of bile duct hyperplasia and centrilobular degeneration in female rats was significantly greater than in controls. A no-observed-adverse-effect level (NOAEL) of 30 ppm DDS in the diet (1.5 mg/kg body weight) was established for rats. DDS was not carcinogenic in these studies.     

Antigenotoxic effect of Saccharomyces cerevisiae on the damage produced in mice fed with aflatoxin B1 contaminated corn

The potential of Saccharomyces cerevisiae (Sc) was evaluated for reducing the micronucleated normochromatic erythrocytes (MNNE) rate in mice fed AFB₁ contaminated corn. The study included two groups fed AFB₁ contaminated corn (0.4 and 0.8 mg/kg), a control fed uncontaminated corn, another group fed uncontaminated corn and 0.3% of Sc (1 × 10⁸ live cells/g), and two groups fed AFB₁ contaminated corn (0.4 and 0.8 mg/kg) plus 0.3% Sc. Weight and MNNE were determined weekly for six weeks. Subsequently, the same determinations were made for another three-week period, but in mice receiving only a normal diet, without AFB₁ and Sc. Results in the first period revealed the following: control and Sc fed mice had similar constant weight increase, and low MNNE rate; mice fed only AFB₁ showed weight decrease and significant MNNE increase; finally, Sc improved weight gain and reduced MNNE produced by AFB₁. In the second period, results exhibited a tendency similar to that of the previous phase in the control and Sc fed mice; the weight and MNNE values improved in the other groups. We also determined the capacity of Sc for adsorbing and modifying the mycotoxin structure. The mixture was filtered to obtain two phases, and AFB₁ content was measured. Sc revealed a potent adsorbent capacity; however, chromatographic determination suggested no structural modification.     

Chronic effects of agar,guar gum,gum arabic,locust-bean gum,or tara gum in F344 rats and B6C3F1 mice

Diets containing 25,000 (2.5%) or 50,000 ppm (5.0%) agar, guar gum, gum arabic, locust-bean gum or tara gum were fed to groups of 50 male and 50 female F344 rats and B6C3F₁ mice for 103 wk. Separate groups of 50 rats and 50 mice of each sex served as controls for each study. There were no significant differences in survival between any of the dosed groups of rats or mice and their respective control groups. Depressions in body-weight gain greater than 10% for dosed groups relative to their respective control groups were observed for male (low dose only) and female mice fed diets containing agar, female mice fed diets containing guar gum (high dose only), male mice fed diets containing locust-bean gum (high dose only) and male and female mice fed diets containing tara gum (high dose only). Depressions in body-weight gain greater than 5% were observed for female rats fed diets containing agar, guar gum or gum arabic. There were no histopathological effects associated with the administration of the test materials. Under the conditions of these bioassays, none of the five polysaccharides was carcinogenic for F344 rats or B6C3F₁ mice of either sex.     

Dietary fat--a requirement for induction of mixed-function oxidase activities in starved-refed rats

Male rats were starved 0-48 hr, and then refed diets containing 0% (F.F.) to 20% corn oil (C.O.) lab chow or 20% coconut oil (C.C.O.) for 1-4 days. Some received phenobarbital sodium (80 mg/kg, i.p. daily) for 1-3 days prior to decapitation. Five cytochrome P-450-dependent indicators were assayed as measures of altered hepatic microsomal function: ethylmorphine N-demethylase (EMDM), N-nitrosodimethylamine (DMN)-N demethylase, aniline hydroxylase (AH), benzo[a]pyrene hydroxylase (AHH) and CO-difference spectra (P-450). Increasing dietary corn oil (0, 0.5, 10, 20%) in control rats resulted in a progressive increase in the activities of these five enzymes. Dietary fat influenced phenobarbital (Pb) inducibility of all mixed-function oxidase (MFO) enzymes measured except AHH. Pb induced the remaining enzymes only 11-22% in animals fed fat-free diet as compared to 119-246% in animals fed coconut oil and corn oil. Rats fed fat-free diet for 21 days without prior food deprivation and administered Pb had 79% more EMDM, 34% more AH and 120% more P-450 than non-induced controls, whereas rats fed 20% corn oil diet had 227% more EMDM, 143% more AH and 128% more P-450. A requirement of dietary fat for induction of MFO by Pb was demonstrated by these starvation-refeeding experiments. Coupled with data recovered from the 21-day studies, these experiments suggest that a compensatory mechanism may be operative during chronic feeding of the fat-free diet to partially return inducibility to the drug-metabolizing system.     

Effects of long-term feeding of ammoniated, aflatoxin-contaminated corn to Fischer 344 rats

The effectiveness of ammonia treatment in reducing the chronic toxicity of aflatoxin-contaminated corn was determined. Fischer 344 rats were fed semi-purified rations containing 20% w/w corn that was either free of aflatoxin or naturally contaminated with 880 micrograms/kg total aflatoxin and was either treated with ammonia gas or was not treated. Therefore the rats that were fed the aflatoxin-contaminated diet received 176 ppb total aflatoxins. Body weight and food consumption were recorded throughout the study; hematological measurements were made after 87 weeks of feeding; and after 91 weeks the rats were killed and histopathological abnormalities were noted. Signs of chronic toxicosis in rats fed aflatoxin-contaminated corn included increased mortality, decreased hematocrit and hemoglobin levels, elevated serum alkaline phosphatase activities, and a 100% incidence of liver neoplasia. These signs did not occur in rats in the other dietary treatment groups, including those fed ammoniated, aflatoxin-contaminated corn. The results provide further evidence that the atmospheric ammoniation process effectively reduces the toxicity of aflatoxin-contaminated corn.