Toxicity of trichothecenes, moniliformin, zearalenone/ol, griseofulvin, patulin, PR toxin and rubratoxin B on protozoan tetrahymena pyriformis.

The inhibitory effects of some fungal products from Fusarium, Trichothecium, Myrothecium and Penicillium were investigated on the protozoan Tetrahymena pyriformis. The dose of mycotoxin which decreased the protozoa growth by 50% in 24 h was defined as inhibitory dose 50 (ID50). The order of toxicity according to the ID50 values were: T-2 toxin greater than trichothecin greater than 4, 15-diacetylverrucarol greater than patulin greater than trichothecolone greater than verrucarol greater than zearalenone greater than PR toxin greater than 3 alpha-acetyldiacetoxyscirpenol greater than zearalenol greater than griseofulvin greater than acetyl T-2 greater than iso T-2 greater than T-2 triol greater than scirpentriol greater than rubratoxin B greater than T-2 tetraol greater than moniliformin. In analogous pairs of trichothecenes their toxicities depended upon the substituents at certain positions of the molecules. Thus, the order of toxicity by the substituents was: at C3 position, H greater than OH greater than OAc [e.g., verrucarol (H at C3) greater than scirpentriol (OH at C3); T-2 toxin (OH at C3) greater than acetyl T-2 (OAc at C3); 4,15-diacetylverrucarol (H at C3) greater than 3 alpha-acetyldiacetoxyscirpenol (OAc at C3)]; at C4 position, OAc greater than OH, and isocrotonyl greater than OH [e.g., acetyl T-2 (OAc at C4) greater than iso T-2 (OH at C4); trichothecin (isocrotonoyl at C4) greater than trichothecolone (OH at C4)]; at C8 position, H greater than isovaleryl greater than OH [e.g., 3 alpha-acetyldiacetoxyscirpenol (H at C8) greater than acetyl T-2 (isovaleryl at C8); T-2 triol isovaleryl at C8) greater than T-2 tetraol (OH at C8); scirpentriol (H at C8) greater than T-2 tetraol (OH at C8)]. Among trichothecenes (without ester groups) with H and OH substituents, the toxicity was inversely related to the number of OH groups in the molecule: verrucarol (2 OHs) greater than scirpentriol (3 OHs) greater than T-2 tetraol (4 OHs). Zearalenone was about 3 times more toxic than its analogue zearalenol. The Tetrahymena cultures exposed 1 d to mycotoxins had protozoa counts/microliters inversely related to doses, and the % transmittance and pH values were directly related to doses.

Toxicity of citreoviridin.

The mycotoxin citreoviridin (CIT) isolated from Penicillium citreoviride was studied to elucidate the mechanism of its toxic actions. In CF#1 mice, near lethal doses of CIT decreased motor activities, body temperature and had cataleptic effects. Male mice appeared to be more susceptible to CIT and had lower subcutaneous (sc) LD50 values and longer CIT-induced hypothermia and hypokinesia. In CIT-treated mice the weights and histology of liver, kidneys and adrenals were normal one week after sc treatment, except for the increased adrenal weights in female mice. Single doses of CIT (sc), given on either day 4 or 5 of pregnancy (perinidation period), had no adverse effect on the rates of pregnancy, implantation of ova and embryonal resorptions in those mice examined on day 12 of pregnancy. CIT (40 mg/kg ip) produced a brief electro-encephalographic (EEG) activation, cardiac sinus arrhythmias and tachypnea in the rabbit. Intravenous (iv) lethal doses of CIT (greater than or equal to 5 mg/kg) caused an EEG activation followed by high voltage delta waves, increased the T wave in the electrocardiogram (ECG) and depressed the respiratory amplitude. The death caused by iv CIT started with the respiratory arrest, followed by isoelectric EEG and ECG was the last to stop. In urethane-anesthetized rabbits CIT decreased the blood pressure, and in succession it lowered, flattened and inverted the T wave of ECG suggesting heart ischemia. These observations indicated that the toxic effects of CIT resulted from respiratory and cardiovascular failures (apnea, delta EEG waves, sinus arrhythmia, hypotension) leading to central nervous system depression due to systemic hypoxia.

Toxic effects of cyclopiazonic acid in the early phase of pregnancy in mice.

The reproductive toxicity of a single oral dose/mouse (15-50 mg/kg) of cyclopiazonic acid (CPA) in the early phase of pregnancy (day 2-8) was investigated. Male mice used in this study were untreated. A limited number of pregnant mice were treated with 66 mg/kg ergonovine maleate (po, sc) to compare its effect with that of an equivalent dose of CPA (50 mg/kg). Among control sperm-positive mice treated with po NaHCO3 solution, 97.5% were gravid on necropsy day (pregnancy day 12). A single dose of CPA (15-50 mg/kg, po) given on days 2 to 8, decreased the pregnancy rates significantly. In groups treated with a single dose of CPA on pregnancy day 4 to 8, vaginal hemorrhage was observed 1 to 7 days after treatment, and it usually resulted in termination of pregnancy (abortion). Fetal resorption rates were higher than the control rate only in the groups treated with 30 mg/kg CPA po on day 4 or 8. CPA decreased body weight gains and the weights of uteri with fetuses. The ovary weights were generally not changed. Ergonovine maleate (66 mg/kg, sc, po) had no significant effect on all of the parameters examined. The estrous cycle returned without any delay in sperm-positive mice in which nidation of fertilized eggs had been inhibited by CPA, and also in nonpregnant mice (used for the LD50 determination) surviving near lethal doses of CPA (50-70 mg/kg, po). The oral LD50 value for CPA in nonpregnant mice was 64 +/- 4.4 mg/kg, and the toxicity signs were ptosis, hypokinesia, hypothermia, action tremor, cessation of food and water intake and resulting cachexia. The duration and intensity of these toxic signs were dose dependent.

Effects of cyclopiazonic acid on the contractility of organs with smooth muscles, and on frog ventricles.

The mycotoxins cyclopiazonic acid (CPA) and ergotamine, and the neurotransmitter serotonin all have the beta-aminoethylindole moiety in common. These compounds enhanced the peristaltic movements of the jejunum, ileum and estrous uterus and produced broncho-constriction in vitro. Atropine and cyproheptadine were able to counter the CPA-induced peristaltic movements of the ileum and jejunum. L-epinephrine was able to stop the contractions induced by CPA on both estrous and pregnant rat uteri. Unlike chlorpromazine, CPA did not block the inotropic effects of dopamine, epinephrine and serotonin in vas deferens. This indicated that the previously reported toxic effects of CPA (hypothermia, catalepsy, hypokinesia, tremor) which resembled the effects of anti-psychotic drugs (chlorpromazine, reserpine) probably were not due to the blocking of the neurotransmitter-receptors. In contrast to ergotamine, which decreased the inotropic effects of serotonin on the uterus, CPA had no anti-serotonin effects. The uterotonic effect of CPA (similar to that of ergotamine) suggested that CPA also might have an adverse effect on the reproductive function of humans and animals consuming CPA-contaminated foods.

Toxicity and neuropharmacology of cyclopiazonic acid.

Cyclopiazonic acid (CPA) was found to have many pharmacological properties in common with the antipsychotic drugs chlorpromazine and reserpine. Thus, in mice CPA at ip doses of 5-14 mg/kg body weight produced hypokinesia, hypothermia, catalepsy, ptosis, sedation without loss of righting reflex, tremor, gait disturbance, dyspnoea, opisthotonus, atypical convulsion and prolonged barbiturate-induced sleep. The ip LD50 of CPA was found to be 13 +/- 0.05 mg/kg. The tremors induced by near-lethal doses of CPA were associated with voluntary or forced movements (action tremors); they worsened during the days following treatment, but they were weak compared with the exhausting and continuous tremors of the whole body caused by 20 mg tremorine/kg (used for comparison). When death occurred only 24-259 min after administration of CPA (11-14 mg/kg), it was preceded by dypsnoea, cyanosis, opisthotonus and clonic leg movements and tonic extension of hind legs (convulsions). When death was delayed (2-6 days after CPA administration), it was preceded by prostration, ptosis, hypothermia, tremor and cessation of food and water intake resulting in cachexia; convulsions were not seen in this group of mice. CPA did not affect the rate of convulsion or death caused by either maximal electroshock or metrazol administration but it did delay the onset of metrazol-induced seizures. In rabbits, 10 mg CPA/kg body weight initially produced tachycardia, tachypnoea and sedation with an activated electroencephalogram. Of three rabbits given 10 mg CPA/kg one died, and in this rabbit slow delta waves were seen just before and during a brief period with clonic leg movements. In this animal death was accompanied by tonic extension of the hind legs, respiratory arrest and cardiac fibrillation; and epileptiform EEG was not seen at any time. The unexpected EEG activation with sedation in rabbits treated with CPA was similar to the effect of reserpine on EEG.

Comparison of the acute toxicities of N-nitrosothiazolidine and N-nitrosomorpholine.

The effects of N-nitrosothiazolidine (NNT) and N-nitrosomorpholine (NNM) on different biological parameters were investigated and compared. The oral LD50 value of NNT (1950 +/- 85 mg/kg) showed that it was about 6 times less toxic than NNM (LD50 = 320 mg/kg, po; Druckrey et al., 1967). Lethal and near-lethal doses (greater than or equal to 1500 mg/kg, po) of NNT caused central nervous system depression (reduced spontaneous motor activity, loss of righting and pain reflexes, without loss of consciousness), stereotypical behavior such as, purposeless chewing jaw movements lasting more than one hour, muscular rigidity, and in some rats, rare and brief clonic convulsions, 3 to 24 h after dosing. These neurotoxic signs, as a whole, were reminiscent of opioid intoxication. Rats that died after NNT-treatment had kidney necrosis in the distal tubules, but all survivors had normal kidneys. NNT (500 and 1000 mg/kg, sc) had no effect on the relative liver weights, but it inhibited liver mitosis at 24, 48, and 72 h after treatment. NNM (100 mg/kg, sc) decreased the relative liver weights on 3 posttreatment days; it inhibited liver mitosis after 24 h and enhanced it after 48 h in male rats. Both NNT and NNM increased the relative adrenal weights, but only NNM enhanced adrenocortical mitosis. In general, NNT had no effect on serum enzymes (SGOT, SGPT, LDH, HBDH), but it increased blood urea nitrogen (BUN) and serum creatinine 24 h after administration. Pretreatment of rats with 3 doses of NNT (150 mg/kg X d, po) increased the pentobarbital-induced sleep (PST) by 26% (not significant), while 3 doses of NNM (50 mg/kg X d, po) increased PST by 188%. In addition, NNM caused a severe centrilobular liver necrosis and glycogen depletion, associated with a marked rise in serum enzymes (SGOT, SGPT, LDH, HBDH) and fall in serum glucose. Compared with NNM, NNT, which was found in fried bacon (Kimoto et al., 1982; Gray et al., 1982), seemed to be a relatively nontoxic nitrosamine.

Comparison of the effects of N-nitrosodimethylamine on pregnant and nonpregnant Holtzman rats.

Single oral doses of N-nitrosodimethylamine or olive oil were given to nonpregnant and pregnant female Holtzman rats on different days of pregnancy (days 7-18, where day 0 was considered to be the sperm positive day). Serological and histopathological studies were performed on animals killed 2 days after treatment. In comparison with the values obtained in nonpregnant controls, the following parameters in pregnant controls were significantly increased: relative liver weights (days 9-20), liver ascorbic acid concentrations (day 12), blood urea nitrogen (days 16-20), serum triglyceride (days 14-20), serum inorganic phosphorus (days 12-18), and serum glutamic-pyruvic transaminase (days 14-20). The following parameters were decreased in pregnant rats compared with nonpregnant controls: relative organ weights (kidneys, adrenals and thyroids), serum glucose (days 12-20), total serum protein (days 9 and 16-20), and serum alkaline phosphatase (day 20). The serum cholesterol levels in pregnant rats were significantly decreased on days 9-15 of pregnancy and significantly increased on day 20. The numbers of mitotic cells in the livers of pregnant rats were greatly increased compared with nonpregnant rats on all days of pregnancy, while the adrenal cortex contained a significantly higher number of mitotic cells only on days 16 and 18. Compared with control values, NDMA given orally (15 or 20 mg/kg body weight) increased the following in both pregnant and nonpregnant rats: numbers of mitotic cells in the liver and adrenal cortex, relative adrenal weights, serum glutamic-oxaloacetic transaminase and serum glutamic-pyruvic transaminase. NDMA treatment decreased liver ascorbic acid and total serum protein in both pregnant and nonpregnant rats. In nonpregnant rats NDMA also increased relative liver weights (not significant) and serum alkaline phosphatase levels. NDMA increased serum alpha-hydroxybutyric dehydrogenase in pregnant rats on day 20 and decreased foetal weights (in rats treated on days 13 and 18). NDMA treatment was not lethal to nonpregnant rats or to pregnant rats up to day 16 of pregnancy, but single oral doses of 15 and 20 mg NDMA/kg killed 9.4 and 35.3%, respectively, of rats treated on day 18 of pregnancy. In general, the acute toxic effect of NDMA, as measured by changes in the above parameters, was greater in pregnant than in nonpregnant rats, especially near the end of pregnancy.

Hepatic effects of R-goitrin in Sprague-Dawley rats.

In male and female rats, R-goitrin treatment increased the relative liver weights; in some of the enlarged livers a high incidence of mitotic figures was observed. R-goitrin treatment increased the relative thyroid weight, more in male rats than in females. The thyroids of R-goitrin treated rats were either hyperplastic or had most follicles filled with pale-staining colloid and rarely follicles with normal, well-stained colloid. At certain doses, R-goitrin increased serum triglycerides, cholesterol, total protein, albumin and calcium, but it decreased serum thyroxine and urea. However, most of these changes in serum chemistry were small. R-goitrin caused a temporary increase in urinary ascorbic acid output in both sexes, but the liver ascorbic acid level was increased only in female rats. The duration of pentobarbital-induced sleep was significantly prolonged by R-goitrin pretreatment only in male rats.

A comparison of ascorbic acid excretion with other indicators of nitrosamine hepatotoxicity.

All of the 4 noncarcinogenic nitrosamines (NA) and 4 of 7 carcinogenic nitrosamines examined increased the urinary ascorbic acid output after oral administration to rats. Of the remaining carcinogenic nitrosamines, dimethyl-NA decreased, and diethyl-NA and methyl-n-pentyl-NA only marginally affected ascorbic acid output. All of the carcinogenic nitrosamines, except dipentyl-NA, increased pentobarbital-induced sleeping time (PST), serum glutamic oxalacetic transaminase (SGOT) and produced loss of glycogen and necrosis in the centrologular area of the liver after 1 or 3 oral doses. In contrast, noncarcinogenic nitrosamines and dipentyl-NA shortened PST and had no effect on liver histology (light microscopy) and SGOT. Generally, changes in ascorbic acid output correlated neither with carcinogenicity nor acute hepatotoxicity of known nitrosamines, hence the ascorbic output could not be used to predict the carcinogenicity of unknown or untested nitrosamines.

Positive inotropic action of Solanaceae glycoalkaloids.

Six glycoalkaloids and one aglycone were tested for cardiotonic activities and compared with K-strophanthoside by use of the isolated frog heart. The decreasing order of potency was as follows: K-strophanthoside greater than tomatine greater than alpha-chaconine=alpha-solanine greater than demissine=commersonine greater than beta-chaconine greater than solanidine. Cardiotonic activity was directly related to the number of sugars in the molecule in which the glycoalkaloids had a common aglycone. On the other hand, glycoalkaloids with dissimilar aglycones, but with identical sugars, such as tomatine and demissine, differed significantly with respect to cardiotonic potency. Apparently, therefore, the cardiotonic activity of glycoalkaloids on the frog heart is determined by the nature of the aglycone and the number of sugars and not by the kinds of sugars or their stereochemical configuration.

Excretion, distribution and metabolic fate of 3H-alpha-chaconine.

3H-alpha-Chaconine was poorly absorbed from the gastrointestinal tract and rapidly excreted in feces when administered orally to male rats. Intraperitoneal administration of low doses (5 to 10 mg/kg) resulted in urinary and fecal excretion of metabolites, and probably involved biliary excretion. High, toxic, intraperitoneal doses (15 to 25 mg/kg) depressed fecal and urinary elimination, and resulted in accumulation of tritium in various tissues. The major metabolite appeared to be the aglycone, solanidine. Alpha-chaconine is very similar to alpha-solanine in its elimination by and distribution in tissues of rats.

Pharmacology and toxicology of chaconine and tomatine.

The pharmacological responses produced by alpha chaconine and tomatine on guinea pig ileum, on the isolated electrically stimulated frog ventricle, and recordings of EEG, ECG, respiration and blood pressure in the rabbit showed no essential differences from those produced by alpha solanine. The LD50 values of chaconine and solanine in the mouse and rabbit are also similar and suggest that compounds other than these are probably responsible for the predominant toxic effects of certain hybrid potatoes in man and animals. The failure of the three glycoalkaloids to produce a significant teratological effect in the chick embryo lends no support to the hypothesis that they may be the teratogens responsible for certain congenital malformations in man.