Marvin A. Friedman

Inhibition of mouse testicular DNA synthesis by mutagens and carcinogens as a potential simple mammalian assay for mutagenesis

Since mutagenic substances induce structural changes in DNA, studies were undertaken to determine whether substances would modify DNA replicative activity. DNA synthesis was quantitated 3.5 h after drug administration as uptake into DNA of a 30-min pulse of 10 muCi of [3H] thymidine. Combinations of methylurea (20000 mg/kg) and sodium nitrite (150 mg/kg) administered p.o. resulted in gastric synthesis of nitrosomethylurea and inhibited testicular DNA synthesis by 83%. Combinations of methylurea and sodium nitrite of 1000 and 100 mg/kg respectively inhibited DNA synthesis by 75%. With dimethylamine and sodium nitrite, a combination which results in gastric synthesis of dimethylnitrosamine, inhibitions of 65 and 57% were observed at 2000 mg/kg together with 150 mg/kg and 1000 mg/kg in combination with 100 mg/kg, respectively. In separate experiments, dimethylnitrosamine (50 mg/kg, p.o.) and diethylnitrosamine (100 mg/kg, i.p.) inhibited thymidine uptake by 30 and 89%, respectively. The mutagenic polynucelar hydrocarbon 3-methylcholanthrene (15 mg/kg) inhibited DNA synthesis by 95% and safrole (640 mg/kg), a mutagenic methylenedioxybenzene derivative, inhibited by 60%. Cadmium chloride (10 mg/kg), acetylaminofluorene (160 mg/kg) and dibutylnitrosamine (500 mg/kg) also induced statistically significant effects. Noncarcinogenic analogues of these substances (anthracene, 125 mg/kg; diphenyl-nitrosamine, 500 mg/kg; piperonyl butoxide, 640 mg/kg, and methylurea, 2000 mg/kg in combination with sodium nitrate) were inactive. Highly toxic substances (potassium cyanide, 2.5 mg/kg; 2,4-dinitrophenol, 20 mg/kg; and lead acetate, 150 mg/kg) were also inactive. This index of mutagenicity appears to have considerable sensitivity and therefore may have potential in drug evaluation.

Induction of micronuclei in mouse and hamster bone-marrow by chemical carcinogens

Four carcinogens, dimethylnitrosamine (DMN), acetylaminofluorene (AAF), aflatoxin B1 (AFB1), and 3-methylcholanthrene (3-MC) were tested acutely and chronically in the micronucleus test in both mice and hamsters. In acute studies using mice, all compounds increased the relative frequency of micronuclei when compared to controls. There was a dose response which tended to tail off as bone-marrow suppression was observed. The order of activity appears to be DMN greater than AFB1 greater than or equal to 3-MC greater than AAF. In acute hamster studies, AFB1 was inactive at the highest dose tested (3 mg/kg). In the case of hamsters, all other compounds increased the incidence of micronuclei with DMN greater than 3-MC greater than AAF. Subacute studies with hamsters revealed that 3-MC was active at 1 week but not 8 weeks while DMN was active at both 1 and 8 weeks. AAF was active at 8 and 12 weeks and AFB1 was active at 12 weeks but not 8 weeks. The authors conclude that this test does not appear useful in chronic protocols and probably more reflects carcinogenicity of the test compounds than acute toxicity. Nevertheless, acute studies demonstrate that this test procedure is of value in the characterization of a compounds relative mutagenic potential.

Protective effects of freeze dried swordfish on methylmercury chloride toxicity in rats

This study quantifies selenium in swordfish and examines its prophylactic effect on methylmercury poisoning in rats. It was found that swordfish have twice as much selenium as mercury, and that a diet which included freeze-dried swordfish protected rats from the toxic effects of methylmercury.

Interactive mutagenicity of sodium nitrite, dimethylamine, methylurea and ethylurea

Groups of mice were treated per os with sodium nitrite either alone or in combination with nitrosatable amino compounds and tested in the host mediated assay. When mice were treated with sodium nitrite in combination with dimethylamine a small(4-fold) but significant increase in mutant frequency (MF) was observed. Ethylurea or methylurea in combination with sodium nitrite induced 10- or 850-fold increases in MF, respectively. The response to methylurea was dose-dependent with a 6- and 30-fold increase in MF at 5.4 and 11.5 mg/kg NaNO2 and a 6-fold increase at 108 mg/kg methylurea. That this response reflected gastric nitrosation was shown by the disappearance of the response if NaNO2 administration preceded methylurea treatment by 10 min. High MFs were observed if NaNO2 was administered 10 or 20 min after methylurea.

Paradoxical effects of piperonyl butoxide on the kinetics of mouse liver microsomal enzyme activity.

Abstract The kinetics of the effects of piperonyl butoxide on mouse liver aminopyrine demethylase, aniline hydroxylase and biphenyl hydroxylases were investigated. Piperonyl butoxide competitively inhibited aminopyrine demethylase in vitro and in vivo, and aniline hydroxylase in vitro; however, in vivo inhibition of aniline hydroxylase was uncompetitive. Contrastingly, in vitro piperonyl butoxide noncompetitively inhibited biphenyl-4-hydroxylase, while competitively stimulating biphenyl-2-hydroxylase. Sulfoxide, a structurally related methylenedioxyphenyl derivative, also noncompetitively inhibited biphenyl-4-hydroxylase and competitively stimulated biphenyl-2-hydroxylase. Furthermore, when microsomal enzyme preparations from control and piperonyl butoxide-treated mice were mixed, aminopyrine demethylase and aniline hydroxylase activities were the same as the control alone, indicating that the inhibitory factors were enzyme bound.

Suppression of dimethylnitrosamine mutagenicity by sodium nitrite

Summary Doses of NaNO2 as low as 25 mg/kg were found to lower the frequency of observed mutants produced by dimethy1nitrosamine (DMN) in S. typhimurium G46 in the host-mediated assay. The degree of reduction of mutant frequency (MIT) was greatest when NaNO2 administration preceded DNIN treatment by 45 min. Sodium nitrite in the diet of mice also lowered the MF of DMN when the animals served as hosts in mutagenicity testing. NaNO2 had no effect on the mutagenicity of 1,3-bis-(β-chloroethyl)-I-nitrosourea (BCNU) which was mutagenic to S. typhimurium both in vitro and in the host-mediated assay. Therefore, the authors conclude that NaNO2 is acting on transformation of DMN to its active form.

Acute lethality of D- and L-ethionine in Swiss mice

The acute, 7-day LD50 in Swiss mice of D-ethionine administered intraperitoneally, was determined as 185 mg/kg with 95% confidence limits of 163 and 210 mg/kg. L-ethionine, the stereoisomer, which is more potent in inhibiting liver RNA synthesis, was not lethal at 2500 mg/kg. This acute toxicity of D-ethionine was suppressed by L-methionine and adenine. Blood accumulation in the thoracic cavity and pericardium was the probable cause of death.

Effects of piperonyl butoxide on dimethylnitrosamine metabolism and toxicity in Swiss mice.

The effects of piperonyl butoxide (PB) on the metabolism and toxicity of dimethylnitrosamine (DMN) in Swiss mice were determined. PB, at doses of 10 and 20 mg/kg, inhibited DMN demethylase 45 min after treatment by 18 and 37%. These inhibitory effects were marked 0.5 hr after PB (640 mg/kg) treatment and reached maximal effects at 2 hr when there was 55% inhibition of enzyme activity. The inhibition by PB continued for 24 hr where enzyme activity was suppressed by 35%. At 48 hr after treatment there was stimulation of enzyme activity. Enzyme kinetic determinations showed no change in Km but Vmax decreased from 129 to 49 mumol CH2O/min-g liver. PB (640 mg/kg) inhibited DMN (500 mg/kg; im) mutagenicity in the host-mediated assay, decreasing the mutant frequency by 42%. Paradoxically, PB (640 mg/kg) had no effect on the alkylation of nucleic acids or proteins in mouse liver, kidney, lung, or spleen. In addition, pretreatment with PB (640 mg/kg) had no effect on the LD50 of DMN.

Suppression of dimethylnitrosamine metabolism and toxicity by nitrososarcosine and other nitrosamines.

Groups of rats were pretreated with nitrososarcosine (NS), diethylnitrosamine (DEN), or dibutylnitrosamine (DBN) in order to observe potential effects on dimethylnitrosamine (DMN) toxicity and metabolism. NS and DEN completely suppressed DMN-induced inhibition of liver protein synthesis while the case with DBN was not as clear. DMN metabolism was effected by suppression of DMN demethylase activity, and an inhibition of RNA, DNA, and protein methylation by [14C]DMN by each nitroso compound.

Suppression of dimethylnitrosamine mutagenicity by nitrososarcosine and other nitrosamines.

Nitrososarcosine, not mutagenic itself in the host-mediated assay using Salmonella typhimurium G46 as indicator organism, lowered the mutant frequency produced by dimethylnitrosamine (DMN). Mutant frequency was significantly depressed when 1.0 g/kg nitrososarcosine was administered by gavage 0.5--2.0 h prior to intramuscular injection of 500 mg/kg DMN. Doses of nitrososarcosine as low as 37.2 mg/kg administered 45 min prior to dimethylnitrosamine treatment produced statistically significant reduction of mutant frequency. Dimethylnitrosamine, diethylnitrosamine and dibutylnitrosamine (500 mg/kg) also partially suppressed the mutant frequency produced by 500 mg/kg DMN when administered 45 min prior to dmn. diethylnitrosamine and dibutylnitrosamine were not found to be mutagenic in this test system.