Kenneth L. Dooley

The Mycotoxin Fumonisin Induces Apoptosis in Cultured Human Cells and in Livers and Kidneys of Rats

Fumonisin B1 is a mycotoxin produced by Fusarium moniliforme, a fungus that infects corn and other grains in the U.S. Fumonisin ingestion causes a variety of effects including equine leukoencephalomalacia and porcine pulmonary edema, and has been associated epidemiologically with human esophageal cancer. Fumonisin B1 produces growth inhibition and increased apoptosis in primary human keratinocyte cultures and in HET-1A cells. In order to set the doses for a 2-year tumor bioassay, male and female F344 rats were fed fumonisin B1 (99, 163, 234, and 484 ppm) for 28 days and the organs examined histologically. There was a dose dependent decrease in liver and kidney weights in the rats. The liver weight loss was accompanied by the induction of apoptosis and hepatocellular and bile duct hyperplasia in both sexes, with the female rats being more responsive at lower doses. The induction of tubular epithelial cell apoptosis was the primary response of the kidneys to dietary fumonisin B1. Apoptosis was present at all doses in the kidneys of the male rats, and occurred in the females only at 163, 234, and 484 ppm fumonisin B1. These results demonstrate that fumonisin B1 treatment causes a similar increase in apoptosis both in vivo and in vitro.

Strain differences in the response of the mouse to diethylstilbestrol

BALB/c StCrlfC3Hf/Nctr, C57BL/6/, C57BL/6 X BALB/c F1 hybrid (B6CF1), and monohybrid-cross offspring from the breeding of B6CF1 mice were examined with respect to uterine, vaginal, and thymus responses to diethylstilbestrol (DES). About 400 mice of each genetic population were used. Weanling mice were fed DES at dietary concentrations of 2.5 to 1,000 ppb (microgram/kg feed) for 6 days and were killed by cervical dislocation about 20 hr after removal of the feed. C57BL/6, B6CF1, and the monohybrid-cross offspring did not differ in the uterine-weight response to DES, but the slope of the dose-response line was shallower for the BALB/c than for the other strains. Dietary DES concentrations of 250 ppb or more inhibited the uterotrophic response in all populations. Vaginal cornification occurred at lower concentrations of DES in the C57BL/6 strain than in the B6CF1 animals. BALB/c and monohybrid-cross offspring were indistinguishable from each other in their vaginal response to Des and were less sensitive to DES than the other mouse populations. The use of ethanol or corn oil as the solvent for mixing DES into the diet had no apparent effect on the uterine weight or vaginal response in any of the mice. DES depressed thymus weight in a dose-related fashion at dietary concentrations of 100 ppb and above in all genetic populations.

High-performance liquid chromatographic analysis of the antituberculosis drugs aconiazide and isoniazid

Reversed-phase HPLC methods are described for determining the stability and concentration certification of the antituberculosis prodrug aconiazide (ACON) in aqueous dosing solution and for assessing the concentrations of ACON and isoniazid (INH) in plasma from ACON-treated male and female Fischer-344 rats. ACON was analyzed in plasma by direct injection; it was separated on a 250 x 4.6 mm I.D. 5 microns C18 column using a 40% aqueous methanol mobile phase containing 5 g/l ammonium formate, and detected at 313 nm. INH was determined in the plasma of treated rats after a two-step precipitation of plasma proteins; it was separated on a 250 mm x 4.6 mm I.D. 5 microns CN column, eluted with 5% aqueous isopropanol containing 5 g/l ammonium formate, and detected with an electrochemical detector at +0.8 V. These methods allow a simple, rapid, and reliable determination of ACON and INH in plasma down to 0.1 micrograms/ml.

Metabolism of 1-nitropyrene in mice: transport across the placenta and mammary tissues

The distribution and metabolism of the environmental pollutant 1-nitropyrene was studied in C57B1/6N mice following oral or intraperitoneal dosing. When administered by gavage, 1-nitropyrene and its metabolites demonstrated biphasic elimination kinetics from the blood, with half-lives of 0.3 and 1.8 days and a distribution volume of 74 ml. Intraperitoneal administration resulted in similar biphasic elimination, with half-lives of 0.5 and 3 days and a distribution volume of 98 ml. Treating pregnant C57B1/6N (C3H sire) mice by gavage resulted in similar absorption and elimination kinetics of 1-nitropyrene and metabolites, except that the distribution volume increased to 123 ml. 1-Nitropyrene and/or its metabolites (0.7% of the administered dose) crossed the placenta and accumulated in the fetuses and amniotic fluid, with both C-oxidized and nitroreduced metabolites being detected. Suckling neonates accumulated 1-nitropyrene and its metabolites when their dams were administered 1-nitropyrene by gavage. Each neonate received approximately 0.1% of the administered dose and demonstrated the presence of both C-oxidized and nitroreduced metabolites. These results demonstrate that this environmental pollutant is capable of crossing the placenta or mammary tissues to expose the offspring to a potentially genotoxic compound.

The Effect of Lifetime Sodium Saccharin Dosing on Mice Initiated with the Carcinogen 2-Acetylaminofluorene

Sodium saccharin has been reported to promote the development of urinary bladder tumors in rats following low doses of several carcinogens. To evaluate the generality of this effect between species, an initiation-promotion study was conducted in mice. Weanling female BALB/c mice were initiated with 200 ppm dietary 2-acetylaminofluorene for 90 days. Following a 2-week period of control diet, saccharin was administered at 0, 0.1, 0.5, 1.0, and 5.0% in the diet for the remainder of the 132-week study. An elevated incidence of persistent bladder transitional cell hyperplasia and a low incidence of urothelial and hepatocellular tumors indicated that these organs achieved an adequate dose of the initiator. However, sodium saccharin dosing did not result in an increased incidence of tumors in either the bladder or liver and is therefore not considered to be a promoter of carcinogenesis at these sites in the mouse. Furthermore, sodium saccharin exhibited a modest inhibitory effect on the rate of development of lymphomas in both initiated and noninitiated animals. Interspecies differences in the bladder tumorigenic effect of sodium saccharin and their association with differences in urinary tract physiology are discussed.

Fourteen-Day, Repeat-Dose Toxicity Evaluation of Aconiazide Administered Orally to Male and Female Fischer 344 Rats

Aconiazide, a hydrazone derivative of isoniazid, has been proposed for the treatment of tuberculosis. As a first step toward assessing the safety of this drug, the effects of a daily oral 14-day treatment on weight gain, pathology, and several hematologic and clinical chemistry parameters were determined in Fischer 344 (F344) rats. Dosage-related changes in body weight were observed and these became significant at dosages of 500 mg aconiazide/kg body weight. Pathologic lesions involved the sciatic nerve, liver, and bone marrow, with the incidence typically becoming significantly elevated at dosages of 500 mg aconiazide/kg body weight. Aconiazide treatment caused statistically significant changes in certain clinical chemistry and hematologic parameters; however, the values of these were still within the normal range reported for rats of a comparable age. The plasma concentrations of aconiazide were dosage-related, tended to be higher in females, and did not increase with repeated dosing.

Six-Month Toxicity Comparison of the Antituberculosis Drugs Aconiazide and Isoniazid in Fischer 344 Rats

Aconiazide, a hydrazone derivative of isoniazid, has been proposed for the treatment of tuberculosis. The toxicity of aconiazide was assessed by treating male and female Fischer 344 (F344) rats daily by gavage for 6 months at doses up to 400 mg/kg body wt. For comparison, the toxicity of isoniazid was determined following treatment in an identical manner at equimolar doses. Aconiazide resulted in only one death during the 6-month experiment, whereas isoniazid caused a significant increase in morbidity and mortality. Each drug induced significant dose-related decreases in body weight in both sexes, and isoniazid caused a significant decrease in liver weight in male rats. Isoniazid also induced centrilobular hepatic necrosis in male rats, a lesion not observed upon aconiazide treatment. Plasma drug levels were ≥10-fold greater in rats administered isoniazid as compared to aconiazide. The higher levels of free drug observed with isoniazid may contribute to greater toxicity of isoniazid compared to aconiazide.

Plasma proteins as early biomarkers of exposure to carcinogenic aromatic amines

Two-dimensional gel electrophoresis (2DG) has been used to study the changes induced in dog plasma polypeptides by the known urinary bladder carcinogens, 4-aminobiphenyl (4-ABP) and 2-naphthylamine (2-NA). Treatment with 3-aminobiphenyl (3-ABP) and 1-naphthylamine (1-NA), both considered to be non-carcinogenic, were used as controls. The purpose of this study was: (1) to determine whether or not changes that occurred in the plasma protein patterns were specific to 4-ABP and/or other related carcinogenic arylamines; (2) to measure the time course in the changes of the major polypeptides during dosing and their resynthesis during a recovery period; and (3) to determine, by microsequencing, the biochemical identity of the affected proteins. The results indicate that only the most potent carcinogen, 4-ABP, had the effect of suppressing the expression of some proteins, while the other aromatic amines caused no discernible change in the 2DG patterns during a 12-week dosing period. The 4-ABP caused dramatic suppression of two sets of proteins. One set of three spots had an apparent molecular weight of 32.5 kDa, and a pI of 5.8-6.0. The major component in this group was identified as the beta-chain of haptoglobin. Expression of this protein decreased markedly during the first 2 weeks of treatment and recovered slowly after dosing stopped. Since haptoglobin functions to bind with free hemoglobin and facilitates its elimination from the blood stream, these results can be rationalized as a consequence of 4-ABP binding to hemoglobin in the erythrocyte, resulting in cell death and hemolysis. The 4-ABP modified hemoglobin then binds to haptoglobin and this tertiary complex is purged from the blood stream, resulting in the disappearance of free haptoglobin. A second set of spots (mol. wt., 65 kDa; pI, 6.5-6.6) disappeared much faster than the haptoglobin, and recovered more quickly. The major protein is about one-fifth the intensity of haptoglobin and appeared to be N-terminally blocked. Internal microsequencing of four fragments obtained from tryptic cleavage of the major spot of this group showed significant similarity to the serum albumin sequence of several species. This spot group is not the major serum albumin spot, however, since the latter is readily identified as the most abundant spot on the plasma map. During the course of this study, several other polypeptides in the 2DG map of dog plasma were identified and are presented here.

Acute oral toxicity of n-2-fluorenylacetamide (2-faa) in several strains of mice.

Summary The oral LD50/7 day of 2-FAA has been determined in both sexes of the following strains of mice: CD-1, C57BL/6Cr, C57BL/6j, and BALB/cj. Symptoms of toxicity included: lethargy, sedation, loss of righting reflex, circling, orange-red urine, bladder concretions and paralysis with all limbs rigidly extended. Deaths peaked between 48 and 72 hr. Particle size was shown to affect the LD50/7 day in CD-1 mice. His-topathological examination of the tissues indicated they were essentially normal. The authors thank S. Poiley for the C57BL/6Cr mice used in this study, and Drs. W. Jacques and C. Frith for the histopathological evaluations.

Estimation of the LD1 and extrapolation of the LD0.1 for five organophosphate pesticides

The oral LD50,s for 5 Organophosphate pesticides have been determined in CD-1 strain male and female mice. The values in mg/kg are: Triohlorfon, 800 and 800; Naled, 409 and 330; Dichlorvos, 139 and 133, GC6506, 23.4 and 17.8; Fospirate, 225 and 263 respectively. Toxicity was greater in males with Fospirate and greater in females with Naled and GC6506. The predicted LD1s and the extrapolated LD0.1s have been determined for the 5 organophosphates from an unbalanced design, loaded heavily toward the lower end of the dose-response curve. It has been shown that the slopes of the curves obtained with 50, 100 and 660 animals are parallel for all compound except Fospirate in the 660 mouse experiments. This is probably related to excessive female deaths in the upper segment of the dose-response curve. Sex dependent lethality was observed with Trichlorfon, Dichlorvos and Fospirate with the males being more susceptible than the females except in the case of Fospirate where there was a reversal at the LD50 with greater susceptibility in the females. The conditions for obtaining accurate results in such experiments have been established. The implications of human exposure to low levels of the environmental pollutants have been discussed.ZusammenfassungDie orale LD50 für 5 Organophosphat-Pesticide wurde für männliche und weibliche CD-1 Mäuse bestimmt. Die Werte in mg/kg sind: für Trichlorfon 800 und 800; für Naled 409 und 330; für Dichlorvos 139 und 133, für GC6506 23,4 und 17,8; und für Fospirate 225 und 263. Die Fospirate Toxicität ist größer in männlichen Mäusen; Naled und GC6506 ist toxischer in weiblichen CD-1 Mäusen. Die vorausgesagten LD0.1s und die extrapolierten LD0.1s wurden bestimmt für die 5 Organophosphate mittels einer unbalanzierten Versuchsanordnung; d. h. dem unteren Teil der Dosis-Wirkungs-Kurve wurden mehr Tiere zugeordnet. Es wurde gezeigt, daß der Verlauf der mit 50, 100 und 660 Tieren erhaltenen Kurven für alle Verbindungen mit Ausnahme von Fospirate im Experiment mit 660 Mäusen parallel ist. Die Fospirate Ausnahme ist vermutlich bedingt durch die hohe Mortalitätsrate der weiblichen Mäuse im oberen Abschnitt der Kurve. Eine geschlechtsabhängige Letalität wurde beobachtet; männliche Mäuse sind empfindlicher gegenüber Trichlorfon, Dichlorvos und Fospirate als Weibchen. Fospirate bewirkte eine größere Letalität in Weibchen in der Nähe der LD50. Die Bedingungen zur Gewinnung akkurater Resultate in solchen Experimenten wurden beschrieben. Die Folgen einer Exponierung von Menschen gegenüber kleinen Mengen von Umwelts-Verbindungen wurden diskutiert.