Study of promutagen biotransformation in the Ames test. III. The role of conjugation with glucuronic acid and sulfate in the modification of mutagenic effect of nitrosomorpholine, diethylnitrosamine and cyclophosphamide

The role of reactions of conjugation with uridine diphosphoglucuronic acid (UDPGA) and with 3-phosphoadenosine-5-phosphosulfate (PAPS) in modification of the mutagenic effect of diethyl nitrosamine (DENA), nitrosomorpholine (NM) and cyclophosphane (CP) was studied by the Ames test. It was shown that adding UDPGA to the activating mixture significantly decreased the level of the mutagenic effect of DENA, NM and CP on bacteria Salmonella typhimurium TA 1950, when S9 and microsomal fractions of rat liver homogenate were used. Adding PAPS to the activating mixture when S9 and cytosole fractions were used, did not affect mutagenic action of DENA on S. typhimurium TA 1950 and TA 1535, enhancing the mutagenic effect of CP on TA 1535, with no such influence on TA 1950. Introduction of PAPS into the activating mixture elevated the mutagenic effect of NM on both bacterial strains using S9 fraction but not cytosole fraction.     

Mutagenicities of the pyrolyzates of peptides and proteins.

Pyrolyzates of 10 peptides, 10 proteins and 5 naturally-occurring materials were tested for mutagenicity in the histidine-requiring mutants Salmonella typhimurium TA98 and TA100. Significant mutagenic activity was detected with pyrolyzates of most of these materials. The pyrolyzates requred a liver microsomal fraction, as representative of mammalian metabolism, for their detection as mutagens. Among the pyrolyzates tested, the highest mutagenic activity was observed with that of a tryptophan-containing peptide. The pyrolyzate of protein obtained from tobacco leaf also showed mutagenicity. The higher the protein content in the leaf the higher the mutagenic activity of the pyrolyzate. Protein in a tobacco leaf may be the principal precursor of mutagens in tobacco-smoke condensate.     

Mutagenicity of aminophenyl and nitrophenyl ethers, sulfides, and disulfides.

The mutagenic activity of several aromatic amines and aromatic nitro compounds related to 4,4'-methylenedianiline towards Salmonella typhymurium tester strains TA100 and TA98 was evaluated. The heteroatomic analogs of 4,4'-methylenedianiline which include aminophenyl and nitrophenyl ethers, sulfides and disulfides were assayed in the presence of rat-liver homogenate. The relative mutagenic response of these analogs indicated the following order of activity, --S-- greater than --O-- greater than --CH2--CH2-- greater than or equal to --S--S--. In both tester strains 4-aminophenylsulfone was inactive with and without microsomal activation. The p-nitrophenyl ethers, sulfides and disulfides were relatively strong mutagens without microsomal activation towards TA100. While 4-nitrophenyldisulfide was found to possess significantly different mutagenic activity than 4-nitrothiophenol in TA98, 4-AMINOPHENYl disulfide has similar mutagenic properties to 4-aminothiophenol in both tester straains TA100 and TA98.     

Mutagenic activity of amino acid pyrolyzates in Salmonella typhimurium TA 98.

Pyrolyzates of 25 amino acids and 5 indole derivatives were tested for mutagenicity in the histidine-requiring mutant Salmonella typhimurium TA 98. Significant mutagenic activity was detected with pyrolyzates of most of the amino acids. These pyrolyzates required a liver microsomal fraction, as representative of mammalian metabolism, to be detected as mutagens. Among the pyrolyzates tested, the highest mutagenic activity was observed with that of L-tryptophan. As little as 10 microgram of the pyrolyzate of L-tryptophan had detectable mutagenic activity toward TA 98. The optimal pyrolysis temperatures for the formation of mutagenic products were shown to be 500 degrees C for L-tryptophan and 600 degrees C for the other amino acids. The results from pyrolyses of some indole derivatives suggest that an amino group at the alpha-position to the carboxyl group of L-tryptophan plays an important role in the formation of mutagens.     

Comparison of the in vitro mutagenicity and metabolism of dimethylnitrosamine and benzo[a]pyrene in tissues from inbred mice treated with phenobarbital, 3-methylcholanthrene or polychlorinated biphenyls.

Homogenates of liver, lung, kidney, stomach, small intestine and colon from 8 strains of mice were compared for their ability to metabolize benzo[a]pyrene (BP) and dimethylnitrosamine (DMN) to mutagens. Females of strains CF1, AKR/J, AU/SsJ, DBA/2J, SWR/J, A/J, C3H/HeJ, and C57BL/6J were either untreated or received phenobarbital (PB), 3-methylcholanthrene (MC) or polychlorinated biphenyls (AR) to induce drug-metabolizing enzymes. The effects of these drugs on organ weight and on the amounts of DNA, S-10 protein, and microsomal protein per unit weight of tissue are reported. Salmonella typhimurium TA92 and TA98 were used as indicators of the formation of mutagens. For each organ there was an optimal balance between amount of tissue homogenate and concentration of test compound for maximal yield of revertants. A sensitive radiometric assay of DMN demethylase (DMND) is described which permits measurement of the enzyme in liver, lung and kidney. DMN at 1 mM is used as substrate. Aryl hydrocarbon hydroxylase (AHH) was measured in all tissue using BP as substrate. AR and MC are very good inducers of AHH activity in livers of mice classified as aromatic hydrocarbon responsive, but not in those classified as hydrocarbon nonresponsive. Responsiveness is strain-specific and genetically regulated. Metabolism of BP to mutagens by liver homogenates was correlated with extent of AHH induction. This dimorphism of response of AHH to inducers was present, but less pronounced, in non-hepatic tissues. Basal activities of AHH and DMND were correlated in livers and lungs from untreated mice. DMND activities were increased less than 2-fold by PB, MC or AR treatments. Metabolism of DMN to mutagens was not closely correlated with DMND activities. Strain of mouse, type of tissue and test substance are important variables in assessing the potential effect of microsomal enzyme-inducing agents on the metabolism of mutagenic substances.     

Absence of mutagenic activity of ticlopidine in the Ames Salmonella/microsome test

Ticlopidine hydrochloride, 5-(o-chlorobenzyl)-4,5,6,7-tetrahydrothieno[3,2-c]pyridine hydrochloride, a platelet aggregation inhibitor, was tested for mutagenic activity in the Ames Salmonella/mammalian microsome test. Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and TA1538 were employed. Two of these strains (TA1535 and TA100) are sensitive to base-pair substitution mutagens, and the remaining 3 are sensitive to frame-shift mutagens. There was no evidence that ticlopidine hydrochloride had any mutagenic activity either in the presence or absence of a liver microsomal supplement.     

Review of the Salmonella typhimurium mutagenicity of benzidine, benzidine analogues, and benzidine-based dyes

We have reviewed the mutagenicity of benzidine analogues (including benzidine-based dyes), with a primary emphasis on evaluating results of the Salmonella/microsome mutagenicity assay. Many of these amines are mutagenic in tester strains TA98 and TA100 but require exogenous mammalian activation (S9) for activity. A few amines with halogen or nitro-groups in the structure are direct-acting mutagens. The addition of a sulfonic acid moiety to the molecule of benzidine reduced the mutagenicity of benzidine; whereas, methoxy, chloro, or methyl group additions did not. Complexation with a metal ion also decreased the mutagenicity. A substitution of an alkyl group on the ortho position next to an amine group also influenced the mutagenicity. Most carcinogenic benzidine analogues are mutagenic, and their metabolism to electrophiles that interact with DNA, leading to mutations, plays a central role in their carcinogenesis.     

Stimulation of serum lecithin-cholesterol acyltransferase activity by phenobarbital in the rat

The activity of serum lecithin-cholesterol acyltransferase was increased on administration of phenobarbital to the rat. This effect was dependent on dose and elapsed time after administration of the drug. Phenobarbital did not stimulate lecithin-cholesterol acyltransferase activity when added to serum from normal animals in vitro. Presumably, phenobarbital increased serum lecithin-cholesterol acyltransferase activity by induction of the microsomal enzyme and subsequent secretion by the liver.     

Mutagenicity of benzo[a]pyrene 7,8-dihydrodiol and 7,12-dimethylbenz[a]anthracene 3,4-dihydrodiol in S. typhimurium mediated by microsomes from rat liver and mouse skin

The mutagenic activities of trans-7,8-dihydro-7,8-dihydroxybenzo[a]-pyrene (BP 7,8-diol) and of trans-3,4-dihydroxy-7,12-dimethylbenz[a]-anthracene (DMBA 3,4-diol) towards S. typhimurium TA100 were measured in assays that were carried out on a micro-scale in liquid medium in the presence of microsomal fractions prepared from mouse skin or rat liver. In the presence of an NADPH-generating system, microsomal enzymes converted both diols into mutagens that were probably the respective 'bay-region' diol-epoxides. The rate of the enzyme-catalysed conversion of the BP 7,8-diol into mutagens by microsomal preparations from mouse epidermis was similar to that occurring with microsomes from rat liver. Pretreatment of mice by the topical application of benz[a]anthracene (BA) or 7,12-dimethylbenz[a]-anthracene (DMBA) increased the mutagenic activity of BP 7,8-diol mediated by mouse skin microsomal preparations by 2-fold and this was paralleled by a 4-fold increase in epidermal aryl hydrocarbon (benzo[a]pyrene) hydroxylase (AHH) activity. The results are discussed in relation to the high susceptibility of mouse skin to polycyclic aromatic hydrocarbon (PAH) carcinogenesis.     

Comparative activation of 3,3'-dichlorobenzidine and related benzidines to mutagens in the Salmonella typhimurium assay by hepatic S9 and microsomes from rats pretreated with different inducers of cytochrome P-450

The metabolic basis of the differential activation of 4 benzidines--3,3'-dichlorobenzidine (DCB), benzidine (BZ), o-tolidine (TOL) and o-dianisidine (DIN)--to mutagens was examined in the Ames test, using Salmonella typhimurium TA98. For each benzidine congener, the comparative activation by 3 rat liver enzyme systems--(i) postmitochondrial supernatant (S9), (ii) S9 + acetylcoenzyme A (S9-Ac) and (iii) microsomes--and the effect thereon of animal pretreatment with 3 cytochrome P-450 inducers--DCB, 3-methylcholanthrene (MC) and phenobarbital (PB)--were examined. DCB was the most activated of the benzidines, with activation by the 3 systems being in the order: S9 = S9-Ac greater than microsomes, whereas dianisidine and tolidine were the least activated. Benzidine was activated only in the S9 systems but the S9-catalyzed activation of benzidine, unlike that of DCB, was enhanced by added acetylcoenzyme A. Pretreatment with either DCB, MC or PB enhanced the activation of DCB, decreased that of benzidine, and had no effect on that of tolidine or dianisidine. The enhanced DCB activation was most pronounced with DCB pretreatment and was 2.5-fold, 2-fold, and 3-fold, in S9-Ac, S9, and microsomes, respectively. The microsomal-catalyzed DCB activation was inhibited by the cytochrome P-450 inhibitors 2,4-dichloro-6-phenylphenoxyethylamine and alpha-naphthoflavone by 93% and 48%, respectively. DCB, but not its congeners, elicited NADPH-dependent metabolite complex formation with microsomal cytochrome P-450. The results show that DCB is the most mutagenic of the 4 benzidines under conditions of cytochrome-P-450-catalyzed activation and suggest that the DCB activation may be catalyzed most effectively by cytochrome P-450 species induced specifically by the compound itself.     

The effect of short-term dietary modification on human fecal mutagenic activity

To assess the effect of short-term modification of diet on human fecal mutagenic activity, 6 subjects consumed 2 dietary regimes hypothesized to affect risk of colorectal cancer. After a 7-day baseline period, a 'low-risk' non-meat diet was consumed for 2 weeks followed by 2 weeks on a 'higher risk' diet which emphasized beef and refined grains. Fecal samples were collected at the end of each diet period and assayed for direct-acting mutagens with the fluctuation test for weak mutagens using Salmonella typhimurium TA100 and TA98 as tester strains. Fecal mutagenic activity on TA100 was increased for all subjects during the 'higher risk' period compared to the 'low risk' period. The average mutagenicity on TA98 was also increased, but the trend was not consistent for all subjects. The baseline diet and non-meat diet resulted in approximately equal mean fecal mutagenicity levels. These findings indicate that a diet high in meat and refined grain, as characterized here, increases fecal mutagenic activity within a 2-week period.     

Mutagenic action of a series of epoxides.

The mutagenicity of a series of 13 epoxide compounds was studied using a bacterial plate assay system. The histidine-dependent tester strains TA98 (for frameshift mutagens) and TA100 (for base-pair substitution mutagens) of Salmonella typhimurium were used. Mutagenicity was evaluated both with and without the additon of rat liver microsomal extract. Dieldrin, diglycidyl ether of bis phenol A and 3 of its homologues were not mutagenic. Allyl glycidyl ether, n-butyl glycidyl ether, vinly cyclohexene diepoxide, glycidol, glycidal-dehyde, diglycidyl ether, diepoxybutane and diglycidyl ether of substituted glycerine were mutagenic in the TA100 strain, causing reversion of the bacteria to histidine independence. Dose-reponse curves of the mutagenicity of the latter 4 compounds were obtained. On a molar basis, glycidaldehyde was about 20-50 times more potent in producing mutation that were the other 3 epoxides in the dose-response test. In general, the mutagenicity of the epoxides was not enhanced or diminished by the addition of microsomal extract.     

Mutagenicity of anti-cancer nitrobenzofuroxans.

Anti-leukaemically active benzofuroxans were tested for mutagenicity in Salmonella typhimurium. Mutagenicity was not found to be correlated to the previously established anti-leukaemic activity. One anti-leukaemically inactive compound after exposure to liver microsomal enzymes proved the most mutagenic of the derivatives for TA100, whereas after similar treatment, the mutagenicity of the most potent anti-leukaemic compound was reduced. All twelve derivatives tested were mutagenic in a base-substitution strain which was defective in excision-repair and also carried a plasmid-linked repair deficiency. Mutagenicity of five dervatives was undetectable in strains proficient for one or the other of the above repair pathways. Nine of the benzofuroxans could also be detected as mutagens in the frameshift tester strain TA98.     

Mutagenicity of fungal metabolites related to aflatoxin biosynthesis.

Fungal metabolites identified as the intermediates in aflatoxin biosynthetic pathway were screened for their mutagenic activity to Salmonella typhimurium TA98. Norsolorinic acid, averufin, and versiconal acetate were found to possess questionable mutagenic activity, but versicolorin A, and sterigmatocystin were significant mutagens relative to aflatoxin B1. The mutagenic activity appears to be related to the bisfuran and not the anthraquinone moiety of the molecule, even though the latter is a key structure of such potent carcinogenic mycotoxin as luteoskyrin.     

Factors affecting mutagenic activity of cigarette smoke condensate in Salmonella typhimurium TA 1538.

Smoke condensates from Burley tobacco, bright-type tobacco and various brands of commercial cigarettes were tested for mutagenicity by using a microsomal test system with Salmonella typhimurium TA 1538. Smoke condensate from Burley tobacco had much higher mutagenic activity than that from bright-type tobacco. Increased mutagenic activity was observed with smoke condensates from Burley tobacco grown with increasing amounts of nitrogen fertilizer, and from commercial cigarettes blended with Burley tobacco. There was a significant correlation between nitrate content of cigarette and mutagenic activity of the resulting smoke condensate. The results suggest that nitrate in cigarettes may influence the formation of potential mutagens during the burning of a cigarette.     

Fecal mutagens from subjects consuming a mixed-western diet

Because of potential significance of fecal mutagens (presumptive carcinogens) in the pathogenesis of colon cancer, feces from 99 healthy subjects from the New York metropolitan area were studied. The diet histories indicate that all participants were consuming a mixed-western diet which is high in total fat and low in fiber. Fecal samples that were incubated under anaerobic conditions at 37 degrees C for 96 h or frozen without incubation, were extracted with hexane: peroxide-free diethyl ether (1:1), partially purified on a silica Sep-pak cartridge and assayed for mutagenicity using the Salmonella typhimurium/mammalian microsome system. Aliquots of fecal samples incubated anaerobically showed a higher frequency of mutagenic activity (per cent samples showing activity) in strains TA98 and TA100 with and without microsomal (S9) activation. In addition, the mutagens requiring S9 activation, were more frequently inactivated when the fecal samples were frozen immediately after defecation and transported to the laboratory. Compared with hexane: ether, extraction of fecal samples with acetone increased the mutagenic activity mostly with TA98 with S9 activation. The HPLC fractionation of hexane: ether extract with methanol: water gradient using reverse phase C-18 column and UV detector at 254 nm indicated that the mutagenic activity (TA98 with S9 activation) is concentrated in several peaks. This is the first demonstration of HPLC profile of fecal samples that are active in TA98 with S9 activation. HPLC profile of fecal extracts and mutagenic activity of these extracts in strains TA98 and TA100 suggest the presence of several types of mutagens in the feces of healthy subjects consuming a high-fat, low-fiber mixed-western diet.     

Preliminary studies on the ability of Drosophila microsomal preparations to activate mutagens and carcinogens

Subcellular fractions from Drosophila melanogaster, known to have several xenobiotic-metabolizing enzymatic activities, were investigated with respect to their ability to biotransform compounds that require metabolic activation before exerting mutagenic effects. Nitrofurazone, dimethylnitrosamine, cyclophosphamide and 2-acetylaminofluorene were activated to mutagens upon incubation with Drosophila microsomes or 20000 x g supernatant: mutagenicity was observed in Chinese hamster ovary cells, Escherichia coli strains 343/113/R-9 and 343/113/uvrB, and Salmonella typhimurium TA1538. Under the conditions used, microsomal preparations of Drosophila were not able to activate benzo[a]pyrene to a mutagen for Salmonella typhimurium TA98. The spectrum of mutagenic effects observed shows some correlation with the known mutagenicity of these compounds in vivo in Drosophila melanogaster. Drosophila microsomes appeared to be at least as active as rat-liver microsomes when compared in this type of mutagenicity testing.     

Studies on the mutagenicity and tumor-initiating activity of methylated fluorenes

Several methylated analogs of fluorene were evaluated as mutagens in Salmonella typhimurium TA98 and TA100 in the presence and absence of microsomal activation. Among the methylated derivatives of fluorene assayed were 9-methylfluorene, 2-fluoro- and 2,7-difluoro-9-methylfluorene, 1,9-, 2,9-, 3,9- and 4,9-dimethylfluorene, 2,3,9-trimethylfluorene and 2,7,9-trimethylfluorene. Mutagenic activity was observed for several of these fluorene derivatives in the presence of rat liver homogenate. The data support a previous observation that a single methyl substituent in the 9-position of fluorene is associated with mutagenic activity within this series of compounds. Substitution with fluorine at both the 2- and 7-positions of 9-methylfluorene was not associated with a loss of mutagenic activity as evidenced by the similar mutagenic activity of 2,7-difluoro-9-methylfluorene and 9-methylfluorene. However, 2,7,9-trimethylfluorene was not mutagenic under these assay conditions. 9-Methylfluorene, 1,9-, 2,9-, 3,9- and 4,9-dimethylfluorene and 2,3,9-trimethylfluorene were active as mutagens in the presence of rat liver homogenate, but were inactive as tumor initiators when assayed on mouse skin.     

Mutagenic activity of furfural in Salmonella typhimurium TA100.

The mutagenic activity of furfural was tested in Salmonella typhimurium strains TA98 and TA100. Furfural produced mutations in the TA100 strain, but not in the TA98 strain. A rat-liver microsomal fraction did not increase the mutagenic activity of furfural in either strain. Mutagenic activity of furfural in the TA100 strain was not increased by benzo[alpha]pyrene in the presence of metabolic activation.     

Modulation of the control of mutagenic metabolites derived from cyclophosphamide and ifosfamide by stimulation of protein kinase A

The phosphorylation of the 2 major phenobarbital-inducible cytochrome P450 isoenzymes IIB1 and IIB2 was increased in intact hepatocytes by the action of the membrane-permeating cAMP derivative N6,O2'-dibutyryl-cAMP. Under these conditions cyclophosphamide and ifosfamide (which are known to be activated by cytochrome P450 IIB1) were investigated for mutagenicity in Salmonella typhimurium TA1535 and TA100 and for cytotoxicity in TA1535. Cyclophosphamide and ifosfamide were transformed to mutagenic and cytotoxic metabolites by the hepatocytes. The activation of both drugs to mutagens was markedly reduced after pretreatment of the hepatocytes with the membrane-permeating cAMP derivative N6,O2'-dibutyryl-cAMP. Cyclophosphamide and ifosfamide activation were reduced to 51% and 38% of unstimulated controls respectively, when hepatocytes were incubated for 1 h with N6,O2'-dibutyryl-cAMP in the presence of the phosphodiesterase inhibitor theophylline, and Salmonella typhimurium TA1535 was used. A marked reduction in mutagenicity of cyclophosphamide (35% compared with unstimulated controls) was also observed under different experimental conditions, namely after pretreatment of the hepatocytes with N6,O2'-dibutyryl-cAMP for 1.5 h without theophylline and using Salmonella typhimurium TA100 as target strain. Continued presence of the cytochrome P450 IIB1 and P450 IIB2 inducer phenobarbital in the stimulation medium increased the mutagenicity of cyclophosphamide and led to an even more marked reduction of mutagenicity by pretreatment of the hepatocytes with N6,O2'-dibutyryl-cAMP and theophylline. In order to investigate whether the observed changes were metabolism-related, the ifosfamide metabolite ifosfamide mustard which does not require metabolic activation by cytochrome P450 was studied under the same conditions. Its mutagenicity was indistinguishable after incubation with N6,O2'-dibutyryl-cAMP-treated or with unstimulated hepatocytes. Also the metabolic formation of cytotoxic metabolites from cyclophosphamide and ifosfamide but not that of ifosfamide mustard was markedly decreased by pretreatment of the hepatocytes with N6,O2'-dibutyryl-cAMP and theophylline. Thus the stimulation of protein kinase A in intact cells has important consequences for the control of genotoxic and cytotoxic metabolites and represents a fast and short-term regulation of it.