Liquid Incubation Assay Research Articles

Quantification of Diazeniumdiolate Mutagenicity in Four Differentin VitroAssays

Diazeniumdiolates are under investigation as possible prodrugs of the multifaceted bioregulatory agent nitric oxide. This study was undertaken to assess further the mutagenic potential of two diazeniumdiolates, DEA/NO (Et2N[N(O)NO]Na) and SPER / NO ( [ H2N ( CH2)3NH ( CH2)4N [ N ( O ) NO−] ( CH2)3NH+3]), which generate NO spontaneously with half-lives at 37°C and pH 7.4 of 2 and 39 min, respectively. The genotoxic potential of these compounds was investigated with the Ames bacterial reverse mutation assay, two mammalian cell gene mutation assays (CHO/HGPRT and L5178Y TK+/−), and an assay for sister chromatid exchange (SCE) using Chinese hamster ovary (CHO) cells. Both diazeniumdiolates had previously been shown to be mutagenic in the AmesSalmonellaplate assay. In the experiments reported here,Salmonella typhimuriumstrain TA1535 was exposed to the compounds in a liquid incubation assay for either 15 min or 48 h without an S-9 fraction. With the 15-min exposure, DEA/NO was mutagenic at concentrations of 0.625 mm(3.5 × control) and greater, while SPER/NO was mutagenic at 0.5 mm(2.7 × control) and above. In the CHO/HGPRT assay, DEA/NO was weakly mutagenic only at the highest concentration used, 20 mm, inducing a mutant frequency per survivor that was 2.5 × control, while SPER/NO was mutagenic at 0.5 mmwith a mutant frequency of 2.5 × control. When the CHO cells were given 10 repetitive 20 mmDEA/NO exposures (3 min each), HGPRT mutant frequency was 4.1 × control. In the L5178Y mouse lymphoma cell TK+/−assay, DEA/NO doubled the mutation rate at 1.82 mm, while SPER/NO's mutation frequency was more than twice that of control at 0.63 mm. DEA/NO was positive in the SCE assay without metabolic activation, yielding significant SCE at 1.25, 2.5, and 5 mmthat was 1.8, 2.2, and 2.6 times control, respectively. SPER/NO increased the SCE by 1.2, 1.4, and 1.3 times at 1.5, 2.0, and 2.5 mm. The results suggest that the two diazeniumdiolates, although mutagenic in the bacteria, are much weaker mutagens in mammalian cells.

Effect of cigarette smoke on the mutagenic activation of various carcinogens in hamster

Male Syrian golden hamsters were exposed for 1 or 2 weeks to smoke produced by commercial non-filter cigarettes for 5 consecutive days in a Hamburg type II smoking machine. Postmitochondrial fractions (S9) prepared from the liver, lungs, and pancreas were used in the Ames liquid incubation assay, in order to assess the effect of cigarette smoke (CS) on the metabolic activation of four groups of procarcinogens. The mutagenic activities of five heterocyclic amines on strain TA98 in the presence of liver S9 mix were induced up to 3.7 times above controls including sham smoke control, while no significant alteration of mutagenicity was observed with 3′-hydroxymethyl- N, N-dimethyl-4-aminoazobenzene and benzo[ a]pyrene on TA98 or with N-nirosobis(2-oxopropyl)amine (BOP) on TA100. A similar stimulation of metabolic activation was also observed for 3-amino-1,4-dimethyl-5 H-pyridol[4,3,- b]indole (Trp-P-1) with S9 from the lungs but not from the pancreas. The mutagenic potential of 11 carcinogens including aflatoxin B 1 (AFB 1) and two other heterocyclic amines was also examined using liver S9 from male hamsters pretreated with phenobarbital (PB) or 3-methylcholanthrene (MC). The numbers of revertant colonies were much higher (2-20-fold) in the presence of MC-treated liver S9 than in the presence of PB-treated liver S9, except in the case of AFB 1 which showed a higher mutagenicity with PB-induced S9. 7,8-Benzoflavone considerably inhibited the activities of 2-amino-3-3-methylimidazo[4,5- f]quinoline (IQ) and Trp-P-1 in the presence of either untreated, MC- or CS-treated liver S9, whereas metyrapone was totally lacking this effect, indicating that cy

Urinary mutagenic activity after different immunosuppressive protocols in renal transplant patients

Cyclosporin (CsA) and azathioprine (AZA) are useful immunosuppressive drugs in the management of kidney and liver transplant recipients. We investigated urinary mutagenicity in three groups of kidney transplant recipients after different immunosuppressive protocols. Urinary mutagenicity was detected in a base-pair strain, E. coli WP2uvrA, in a liquid incubation assay. No mutagenic activity was detected in the urines of patients treated with CsA (4.5 mg/kg); 85% of the urines in the second group treated with AZA (1.26 mg/kg) showed high mutagenic activity, whereas mutagenic activity was found in 40% of the urines of subjects treated with CsA and AZA (3.89 mg/kg + 1.15 mg/kg). These data suggest that immunosuppressive therapy with AZA carries a high risk of urinary mutagenicity, while immunosuppressive combined treatment with CsA and AZA significantly reduces this risk.

N-Benzylimidazole, a potent inducer of rat liver enzymes involved in mutagenic activation of various carcinogens

The effects of N-benzylimidazole (BI) on mutagenic activation of six groups of carginogens were studied with the Ames liquid incubation assay. Intragastric (i.g.) treatment of male Wistar rats for 3 days with BI produced an over 3-fold induction of hepatic microsomal cytochrome P-450 (CYP). The mutagenic activities of five N-nitrosamines on S. typhimurium TA100 and two arylamines on TA98 were induced up to 5 times above controls and much higher inductions on strain TA98 were observed with aflatoxin B 1, two polycyclic hydrocarbons, two aminoazo compounds and six amino acid pyrolysates, which are all known to be metabolically activated by CYP I and/or II species. Compared with the reported results for rats treated with polychlorobiphenyls (PCB), the numbers of revertant colonies were the same or higher (0.7–1.8-fold), indicating that BI and PCB show a similar pattern of induction of CYP-dependent properties. However, intraperitoneally treated liver S9 was inferior to the i.g. treated S9 for all mutagenic activations of 18 carcinogens. In conclusion, these results demonstrate the use of BI for metabolic induction to be an effective alternative for induction with either PCB or a combination of phenobarbital and β-naphthoflavone.

Substances in human urine that strongly inhibit bacterial mutagenicity of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and related heterocyclic amines.

Extracts of human urine were shown to contain substances that strongly inhibited the liver S9-mediated mutagenicity of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in Salmonella typhimurium TA98 strain in a liquid incubation assay. The inhibitory effect was unrelated to cytotoxicity and was similar with urine extracts from smokers and non-smokers. Under similar assay conditions, the mutagenicity of the related amino-imidazoazaarenes, 2-amino-3-methyl-imidazo[4,5-f]quinoline, 2-amino-3,8-dimethylimidazo[4,5-f]-quinoline and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline was also found to be strongly inhibited by urine extracts. Decreased or enhanced mutagenicity was seen with 2-acetyl-aminofluorene and 2-aminoanthracene depending on the type of assay, and the time of incubation in liquid medium. A weak inhibition of the mutagenicity of 2-nitrofluorene, a direct-acting mutagen, was observed only after a short incubation time. Mutagenicity of 4-nitroquinoline N-oxide was not altered by the presence of urine extracts at concentrations shown to be inhibitory for the mutagenicity of heterocyclic aromatic amines. Our data suggest that the inhibitory substances in urine act through their capacity to non covalently bind the parent heterocyclic and aromatic amines, thus affecting their availability in aqueous medium for diffusion into liver microsomes where metabolic activation takes place.

Effects of norharman on the mutagenicity of chlorophenylhydroxylamine and its metabolism with rat liver S9

o,p-Chlorophenylhydroxylamines (CPHAs) (10468-16-3, 823-86-9) only demonstrated mutagenicity in the presence of S9 mix and norharman (NOH) (244-63-3), as well as chloronitrobenzenes. The mutagenic activity of o-CPHA was 30 times higher than that of p-CPHA. When o-CPHA was preincubated with S9 mix without NOH, the mutagenic activity disappeared rapidly. The decrease in activity during the preincubation was suppressed by addition of NOH. HPLC analysis revealed that o-CPHA was metabolized to o-chloroaniline ( o-CA) (95-51-2) and that the metabolic reduction was inhibited by NOH. When microsomes containign NADPH were used instead of S9 mix, o-CPHA exhibited only very weak mutagenicity. The activity in the microsome system, however, was greatly enhanced by adding cytosol or ascorbic acid (50-81-7). These phenomena were only observed in the conventional plate incorporation method. In the case of the liquid incubation assay, in which test compound metabolism and tester strain mutation only occur in the liquid incubation medium, the mutagenic activity of o-CPHA in the microsome system with NOH was comparable to that in the S9 system, indicating that o-CPHA was activated by an enzyme in microsomes in the presence of NOH. Consequently, it was concluded that NOH not only affects the metabolic inactivation of o-CPHA to o-CA by S9, but also the metabolic activation of o-CPHA by microsomes. No appreciable enhancing effects of cytosol and ascorbic acid were observed in the liquid incubation assay, suggesting that these factors affect the stability of CPHA or an active metabolite. The microsome activation of o-CPHA was dependent on NADPH and oxygen; SKF-525A (62-68-0), metyrapone (54-36-4) and α-naphthoflavone (604-59-1) inhibited the mutagenic activity by about 50%, suggesting that cytochrome P-450 was involved in the metabolic activation.

Activation of carcinogenic N-nitrosopropylamines to mutagens by lung and pancreas S9 fractions from various animal species and man

The mutagenic potential of 7 carcinogenic N-nitrosopropylamines was examined by the Ames liquid incubation assay, using lung and pancreas 9000 × g supernatant (S9) fractions from rats, hamsters, mice, rabbits, monkeys and humans for metabolic activation. N-Nitroso(2-hydroxypropyl)(2-oxopropyl)amine (HPOP), N-nitrosobis(2-oxopropyl)amine (BOP) and N-nitrosomethyl(2-oxopropyl)amine (MOP) showed positive mutagenicity in strain TA100 in the presence of lung S9 from each of the uninduced animals and humans. Besides the 3 N-nitrosopropylamines, N-nitrosomethyl(2-hydroxypropyl)amine (MHP) was also positive in the presence of lung S9 from polychlorinated biphenyl (PCB)-induced rats, hamsters and mice. On the other hand, in the presence of pancreas S9 from uninduced or PCB-induced animals, only HPOP and BOP showed positive mutagenicity. In contrast, N-nitrosobis(2-hydroxypropyl)amine (BHP), N-nitrosobis (2-acetoxypropyl)amine (BAP) and N-nitroso-2,6-dimethylmorpholine (NDMM) showed negative mutagenicity in the presence of lung and pancreas S9 from either uninduced or PCB-induced animals and humans. HPOP was a direct-acting mutagen, and lung and pancreas S9 from 5 animal species and man did not affect the activity. BOP was mutagenic even in the presence of bovine serum albumin. The mutagenic activation of MHP by lung S9 from PCB-induced rats, hamsters and mice was completely inhibited by preincubation in an atmosphere of carbon monoxide or by addition of cytochrome c or metyrapone to the S9 mixture, whereas 7,8-benzoflavone totally lacked this effect. However, that of MOP was insensitive to these inhibitors. These results of mutagenicity assay indicate that only the methyl derivatives of N-nitrosopropylamines, MHP and MOP are activated by the lung from 5 animal species and man, whereas the pancreas from all the tested animals did not activate the 7 N-nitrosopropylamines to mutagens, and that the phenobarbital-inducible major cytochrome P-450 in the lung of rodents is involved in the mutagenic activation of MHP.

The kinetics of mutagen excretion in the urine of cigarette smokers

The excretion of mutagens in the urine of cigarette smokers was studied as a model for absorption and elimination of complex carcinogenic and mutagenic mixtures in humans. Urine was collected from an occasional smoker who smoked 1 cigarette (17 mg tar/cigarette) and from a heavy smoker (smokes approximately 20 cigarettes/day) who quit for 2 days and then resumed smoking. Urine samples were collected for 6 days, including a 2-day pre-smoking period for the occasional smoker and pre-abstention period for the heavy smoker, respectively. Mutagen excretion patterns were determined by extracting the mutagens in each urine sample with XAD-2 resin and testing the extract in a microsuspension modification of the Salmonella/microsome liquid-incubation assay using bacterial strain TA98 with metabolic activation. Peak mutagenic activity of the urine collected from the two smokers appeared 4–5 h after the beginning of smoking. Activity decreased to pre-smoking ‘baseline’ levels in approximately 12 h for the occasional smoker, and the activity for the heavy smoker approached the occasional smoker's ‘baseline’ in approximately 18 h after the cessation of smoking. The mutagen excretion patterns of the occasional smoker after smoking a single cigarette suggests that, the mutagens, as detected by the Salmonella assay, are absorbed rapidly (3–5 h) and are eliminated from the body following first order kinetics. The excretion rate constant for the occasional smoker was approximately 0.1 h −1 and the half-life ( T 1 2 ) was approximately 7 h.

Inhibitory effect of organic solvents on the mutagenicity of N-nitrosodialkylamines in Salmonella

The influence of organic solvents on the mutagenicity of 11 N-nitrosamines was examined in Salmonella typhimurium TA100 using the Ames's liquid incubation assay in the presence of rat-liver S9. The mutagenic activities of N-nitrosodimethylamine, N-nitrosodiethylamine, 6 oxidative derivatives of N-nitrosopropylamine and N-nitroso-2,6-dimethylmorpholine were considerably decreased by addition of dimethyl sulfoxide, dimethyl formamide, acetone, 95% ethanol or acetonitrile, which are recommended for use as solvents in the assay by Ames's group, to the incubation mixture. The mutagenic activities of N-nitrosodipropylamine and N-nitrosodibutylamine, which are barely soluble in water, were also suppressed by increasing concentrations of dimethyl sulfoxide. These organic solvents did not appear to exert their influence by desmutagenic and antimutagenic actions. In contrast, the recoveries of unmetabolized carcinogens from preincubation mixtures and from agar plates were significantly higher in the presence of organic solvents than in their absence. The results indicate that the inhibitory effect is a result of interference with the process of metabolic activation by liver S9.

Mutagenic activation of carcinogenic N-nitrosopropylamines by rat liver: evidence for a cytochrome P-450 dependent reaction.

Mutagenic potential of carcinogenic N-nitrosopropylamines was examined by the Ames's liquid incubation assay, using rat liver 9000 g supernatant (S9) fraction for metabolic activation. N-Nitrosobis(2-hydroxypropyl)amine, N-nitroso(2-hydroxypropyl)-(2-oxopropyl)amine (HPOP), N-nitrosobis(2-oxopropyl)amine (BOP), N-nitrosobis(2-acetoxypropyl)amine, N-nitroso-2,6-dimethylmorpholine, N-nitrosomethyl-(2-hydroxypropyl)amine and N-nitrosomethyl(2-oxopropyl)amine all showed positive mutagenicity in strain TA100 in the presence of liver S9 while being negative in strain TA98. With the exception of HPOP and BOP, which were also mutagenic in TA100 without S9 metabolic activation, these N-nitrosopropylamines required the presence of microsomes as a source of enzymes as well as NADP+ as a cofactor for mutagenic activation. Treatment of rats with polychlorinated biphenyls or phenobarbital (PB) resulted in a marked increase in the ability of S9 to activate the seven N-nitrosamines tested whereas 3-methylcholanthrene (3-MC) induction was not effective. All the mutagenic activities were considerably decreased by preincubation in an atmosphere of either carbon monoxide or nitrogen gas or by adding cytochrome c to the S9 mixture. Metyrapone, a specific inhibitor of PB-inducible major cytochrome P-450, considerably inhibited mutagenicity, whereas 7,8-benzoflavone, a specific inhibitor of 3-MC-inducible major cytochrome P-448, was totally lacking this effect. These results demonstrate a correlation between rat liver S9 dependent mutagenicity of six N-nitrosopropylamines and their known carcinogenicity in rat in vivo experiments, and that the PB-inducible major cytochrome P-450 is involved in the mutagenic activation. BOP was also shown to be activated by extrahepatic (lung, kidney, pancreas) tissue S9, blood S9 and bovine serum albumin (BSA) to the extent of 50% of that activity obtained with liver S9. A possible mechanism of BSA-mediated activation of BOP is discussed.

DNA-damaging activity in ethanol-soluble fractions of feces from New Zealand groups at varying risks of colorectal cancer.

Using repair-proficient and repair-deficient strains of E. coli, we investigated the application of a liquid incubation assay to measure the DNA-damaging activity of ethanol-soluble fecal extracts. This method appears to be suitable for the study of a wide range of sample types. It was used to measure the DNA-modifying activity of ethanol-soluble fecal extracts from a group of European colorectal cancer patients. Data were compared with those from Europeans of similar age and sex distribution who did not have bowel cancer. We also studied groups of Maoris, Samoans, and European Seventh-Day Adventists who followed an ovo-lacto vegetarian diet. There are significant levels of DNA-modifying materials in the feces of many Europeans on a mixed diet, regardless of whether or not they have cancer. The number of positive samples was less in the Polynesian groups, and there were no samples that could be unequivocally scored as positive in the Seventh-Day Adventist groups. We conclude that diet can significantly reduce the level of ethanol-soluble mutagens, at least in New Zealand Europeans. The data may provide an explanation for the reduced incidence of bowel cancer in Seventh-Day Adventist groups.

Non-mutagenicity of 27 aliphatic acrylate esters in the Salmonella-microsome test

The mutagenicity of 27 acrylate esters was assessed in the Salmonella-microsome assay. Methyl, ethyl, butyl, t-butyl, pentyl, neopentyl, hexyl acrylate and methacrylate and 2-hydroxyethyl methacrylate were tested; furthermore ethanediol, butanediol, pentanediol, neopentanediol, hexanediol and diethyleneglycol diacrylate and dimethacrylate. None of these 27 acrylate esters appeared to be mutagenic in the standard Ames assay with TA1535, TA1537, TA1538, TA98 and TA100, both with and without Aroclor 1254 or phenobarbital-induced S9 mix. A liquid incubation assay of methyl methacrylate, methyl, butyl and hexyl acrylate with TA100 neither gave any indication of a mutagenic activity of these compounds.

α-chloroepoxides. 2. Mutagenicity of 1-chlorocyclohexene oxide and its thermal isomerization product, 2-chlorocyclohexanone

The pseudo-first-order hydrolysis rate constants in pH 7.4 buffer-acetone solution are reported for 1-chlorocyclohexene oxide and 2-chlorocyclohexanone at 0, 25 and 37°C. The rate constants, in conjunction with product studies, demonstrate that the hydrolysis of 1-chlorocyclohexane oxide quantitatively affords 2-hydroxycyclohexanone and that there is no significant isomerization of 1-chlorocyclohexene oxide to 2-chlorocyclohexanone during the hydrolysis. Both 1-chlorocyclohexene oxide and 2-chlorocyclohexanone were reacted with 2-aminopyridine, a model for adenine, to yield the same product, N-(2′-pyridyl)-2-aminocyclohexanone. The mutagenicity of 1-chlorocyclohexene oxide and 2-chlorocyclohexanone in the Ames liquid incubation assay using TA100 shows 2-chlorocyclohexanone to be slightly more active than 1-chlorocyclohexene oxide, in spite of the finding that 1-chlorocyclohexene oxide is clearly a more reactive electrophile than 2-chlorocyclohexanone. These results are interpreted to indicate the important role that hydrolysis (detoxification) plays in the in vitro evaluation of the proposed ultimate electrophilic metabolites of chloroolefins.

Mutagenicity of N-nitrosobis(2-hydroxypropyl)amine and its related compounds in the presence of rat lung and liver S9.

The mutagenic activities of N-nitrosobis(2-hydroxypropyl)amine (BHP) and its related compounds were studied in Salmonella typhimurium TA100 and TA98 strains by Ames's liquid incubation assay in the presence or absence of lung and liver S9 of rats treated with polychlorinated biphenyl (PCB). BHP and its related compounds, N-nitroso-(2-hydroxypropyl)(2-oxopropyl)amine (HPOP), N-nitrosobis(2-oxopropyl)amine (BOP), N-nitrosobis(2-acetoxypropyl)amine (BAP), and N-nitroso-2,6-dimethylmorpholine (NDMM) showed negative mutagenicity in the absence of lung and liver S9 in TA100 and TA98 strains while those compounds showed positive in the presence of liver S9 in TA100 strain. HPOP and BOP showed positive mutagenic activity in the presence of lung S9 in TA100 strain. HPOP showed the strongest mutagenic activity in the presence of lung and liver S9.

Superoxide dismutase protects against paraquat-mediated dioxygen toxicity and mutagenicity: studies in Salmonella typhimurium.

Paraquat is univalently reduced to the relatively stable, but oxygen-sensitive, paraquat radical (PQ.+). This PQ.+ can react with dioxygen to generate the superoxide radical, which can further generate other more deleterious species of oxygen free radicals (i.e., hydroxyl radical, OH.). These oxygen free radicals are known to cause chromosomal breaks; therefore, it was logical to postulate that paraquat is a mutagen. This proved to be the case when tested in a modified Ames test using a liquid incubation assay. Salmonella typhimurium strains TA98 and TA100 were grown in the presence of various concentrations of PQ, as well as in the presence of known mutagenic compounds: mitomycin C, azide, and proflavine. Paraquat was much more toxic and mutagenic in a simple nutritionally restricted medium than in a rich complex medium and these toxic and mutagenic effects were oxygen dependent. Furthermore, cells containing high levels of superoxide dismutase were more resistant to the toxic and mutagenic effects of paraquat than were cells containing a normal level of this enzyme.