Synthesis and mutagenicity of 3, 3'-dihalogenated benzidines

3, 3'- Difluorobenzidine (F₂ Bz), and 3, 3'- dibromobenzidine(Br₂Bz) were synthesized and compared with 3, 3'-dichloro-benzidine(C1₂ Bz) for ability to revert Salmonella typhimurium. The relativemutagenicities in all systems are CI₂Bz = Br₂Bz > F₂Bz >Bz. F₂Bz, CI₂Bz, and Br₂Bz are direct-acting mutagens towardsS. typhimurium strain TA98. The acetylase-deficient derivativeTA98/1, 8-DNP₆ displays no resistance to induction of mutagenesisby these compounds, in the absence of mammalian activation.With addition of hamster hepatic S-9 activation the mutagenicityof these compounds increases greatly. TA98/1, 8-DNP₆ shows someresistance to this mutagenicity. Multiple Mechanisms must existfor the genotoxicity of 3, 3'-dihalogenated benzidines.     

N-nitrosophenacetin: its synthesis, characterization, mutagenicity, and teratogenicity

Reaction of phenacetin (CAS: 62-44-2; p-acetophenetidide) with nitrous fumes (N2O3) in glacial acetic acid at 0-5 degrees C yields N-nitrosophenacetin (NP), 2-nitrophenacetin, N-nitroso-2-nitrophenacetin (NNP), and other compounds. Both NP and NNP are fairly stable at low temperature (-30 degrees C) but extremely labile at ambient temperature. NP (median lethal dose to Sprague-Dawley rat: 21 mg/kg body wt) is 80 times more toxic than its parent compound phenacetin and is directly mutagenic to bacterial cells including Salmonella typhimurium and Sarcina lutea. The mutagenicity of NP is comparable to that of N-methyl-N'-nitro-N-nitrosoguanidine [(MNNG) CAS: 70-25-7; 1-methyl-3-nitro-1-nitrosoguanidine] and requires no microsomal metabolic activation. The teratogenic potential of NP was studied in White Leghorn chick embryos given a single dose of 5-15 micrograms/egg on day 6 of incubation. A low incidence of exencephaly and eyelid defect and a high incidence of feather and claw malformations were found in the treated group; no such malformed embryos were found in the control group. The teratogenicity of NP was found to be weaker than that of MNNG, but stronger than that of N-methyl-N-nitrosourea (CAS: 684-93-5), dimethylnitrosamine (CAS: 62-75-9; N-nitrosodimethylamine), and diethylnitrosamine (CAS: 15-18-5; N-nitrosodiethylamine).     

Synthesis, purification and mutagenicity of 2-hydroxyamino-3-methylimidazolo[4,5-f] quinoline

Synthesis of 2-hydroxyamino-3-methylimidazolo [4,5-f]quinoline(N-hydroxy-IQ), a reactive metabolite of 2-amino-3-methylimidazolo[4,5-f]quinoline(IQ), was achieved by a modification of an earlier method. N-Hydroxy-IQwas purified by a two-step procedure involving C₁₈ Sep-Packand semi-preparative h.p.l.c. Additional h.p.l.c. methods weredeveloped to monitor the synthesis of N-hydroxy-IQ, and to measureIQ and other IQ derivatives on the same h.p.l.c. profile. Thestructure of N-hydroxy-IQ was confirmed by mass spectral analysisfollowing derivatization to azoxy-IQ, phenyl-azoxy-IQ and acetoxy-acetamido-IQ,and by chemical reactivity studies. Mutagenicity studies withthe nitroreductase-deficient strain of Salmonella TA98 showedthat N-hydroxy-IQ is directly mutagenic, having a specific activityof 2 x 10⁴ revertants/nmol. The data confirm that N-hydroxy-IQis a mutagenic metabolite of IQ and further implicate the hydroxylaminein the carcinogenkity of IQ.     

Synthesis of 6-methylchrysene-l,2-diol-3,4-epoxides and comparison of their mutagenicity to 5-methylchrysene-l,2-diol-3,4-epoxides

The syn- and anti-isomers of l,2-dihydroxy-3,4-epoxy-l,2,3,4-tetrahydro-6-methykhrysene(6-MeC-l,2-diol-3,4-epoxide) were synthesized and their mutagenkactivities in Salmonella typhimurium were compared with thoseof the syn- and anti-isomers of l,2-dihydroxy-3,4-epoxy-l,2,3,4-tetrahydro-5-methykhrysene(5-MeC-l,2-diol-3,4-epoxide). The most mu-tagenic compound wasanti-5-MeC-l,2-diol-3,4-epoxide, followed by syn-5-MeC-l,2-diol-3,4-epoxide.At the same doses, neither of the 6-MeC-l, 2-dioI-3, 4-poxideswas mutagenic. These results demonstrate the enhancing effecton mutagenicity of a methyl group in the same bay region asthe epoxide ring of a diol epoxide.     

The mutagenicity and DNA base sequence changes induced by 1-nitroso- and 1-nitropyrene in the cl gene of lambda prophage

The mutagenicity of 1-nitropyrene and its reduced metabolite1-nitrosopyrene was determined in the lambda cI gene of an Escherichiacoli uvr^- lysogen. 1-Nitropyrene induced a mutation frequencyof 3.8 x 10^–6, which was {small tilde} 2-fold higher thanthe background mutation frequency, whereas an equimolar doseof 1-nitrosopyrene induced a much higher mutation frequencyof 1.4 x 10^–4. Previous studies have established thatboth compounds form the same premutagenic lesion, viz. N-(deoxyguanosin-8-yl)-l-aminopyrenein bacterial DNA. In order to determine how this initial premutationallesion is converted to a stable heritable mutation, DNA sequenceswere determined for 30 mutations induced by 1-nitrosopyrenethat mapped between bp 1 and 352 in the lambda cI gene of E.colilysogens. We show here that these mutations are mainly frameshiftsinvolving the addition or deletion of a single GC or CG basepair. A small proportion of mutations were base substitutionswhich were equally divided between transitions and transversions.These also occurred primarily at GC or CG sites.     

The mutagenicity and DNA base sequence changes induced by 1-nitroso- and 1-nitropyrene in the cI gene of lambda prophage

The mutagenicity of 1-nitropyrene and its reduced metabolite 1-nitrosopyrene was determined in the lambda cI gene of an Escherichia coli uvr- lysogen. 1-Nitropyrene induced a mutation frequency of 3.8 x 10(-6), which was approximately 2-fold higher than the background mutation frequency, whereas an equimolar dose of 1-nitrosopyrene induced a much higher mutation frequency of 1.4 x 10(-4). Previous studies have established that both compounds form the same premutagenic lesion, viz. N-(deoxyguanosin-8-yl)-1-aminopyrene in bacterial DNA. In order to determine how this initial premutational lesion is converted to a stable heritable mutation, DNA sequences were determined for 30 mutations induced by 1-nitrosopyrene that mapped between bp 1 and 352 in the lambda cI gene of E.coli lysogens. We show here that these mutations are mainly frameshifts involving the addition or deletion of a single GC or CG base pair. A small proportion of mutations were base substitutions which were equally divided between transitions and transversions. These also occurred primarily at GC or CG sites.     

Induction of hyperplastic liver nodules in Wistar and MRC-Wistar rats by phenobarbital and the liver carcinogens acetoxime, 1-nitroso-5,6-dihydrouracil and 3-nitroso-2-oxazolidinone

We tested the ability of phenobarbital and two liver carcinogens, acetoxime and 1-nitroso-5,6-dihydrouracil (NDHU), to induce hyperplastic liver nodules (HLN) in MRC-Wistar and Wistar rats, using a system that included a single diethylnitrosamine (DEN) treatment, partial hepatectomy, and administration of the test compound in drinking water for 8 weeks. All three compounds induced significant HLN frequencies (number of HLN/cm2) in both rat strains. When the results for each strain were "normalized" for each compound and then combined, HLN frequency in MRC-Wistar rats was significantly lower (P less than 0.01) than that in Wistar rats. The weak liver carcinogen 3-nitroso-2-oxazolidinone (NOZ) did not induce a significant HLN frequency in MRC-Wistar rats. Acetoxime was highly volatile and was not mutagenic in the Ames test under a variety of conditions. The results for acetoxime are of interest because simple oximes are common constituents of oil paints. HLN induction by nitrosodihydrouracil is of interest because, unlike most liver carcinogens, this compound probably does not require metabolic activation and shows only a mild acute hepatoxicity.     

Synthesis and mutagenicity of 5-alkyl-substituted chrysene-1,2-diol-3,4-epoxides

In order to explore the relationship between structure and mutagenicityof bay region diol-epoxides of chrysene substituted with analkyl group in the bay region, we compared the mutagenicityin Salmonella typhimurium TA 100of anti-1,2-dihydroxy-3,4-epoxy-1,2,3,4-tetrahydrochrysenewith its 5-methyl, 5-ethyl and 5-propyl derivatives. The resultsshowed that anti-l,2-dihydroxy-3,4-epoxy-l,2,3,4-tetrahydro-5-methylchrysene(7400 revertants/nmol) was the most mutagenic of these diol-epoxidesfollowed by anti-1,2-dihydroxy-3,4-epoxy-1,2,3,4-tetrahydrochryseneand its 5-ethyl derivative (1100 revertants/nmol). The 5-propylsubstituted diol-epoxide was inactive at the doses tested. Theresults demonstrate that steric factors are dominant in theexpression of methylchrysene diol-epoxide mutagenicity in S.typhimuriumand suggest that the molecular shape of the 5-methyl substituteddiol-epoxide leads to a unique reaction with DNA associatedwith high mutagenicity and tumorigenicity.     

Similar carcinogenic effects in rats of 1-ethyl-1-nitroso-3-hydroxyethylurea and 1-hydroxyethyl-1-nitroso-3-ethylurea

The two isomeric N-nitroso derivatives of the dialkylurea, 1-ethyl-3-(2-hydroxyethyl)urea,were given by gavage to 20 male F344 rats for 30 weeks at equimolardoses. The tumorigenic responses were compared with those toa similar dose of nitrosoethylurea or nitroso-2-hydroxyethylurea.Each of the nitrosomonoalkylureas caused death from tumors morerapidly than the analogous nitrosodialkylurea. Each of the nitrosodialkylureasinduced a broader spectrum of tumors in the rats than did eithernitrosoethylurea or nitroso-2-hydroxyethylurea, including neoplasmsof the thyroid, lung, skin, colon, mesotheliomas and neoplasmsof the brain and liver in high incidence, the last two of whichwere not seen in animals given the nitrosomonoalkylureas. Onthe other hand, there were fewer tumors of the forestomach inrats given the nitrosodialkylureas than with the nitrosomo-noalkylureas.The major difference between 1-nitroso-1-eth-yl-3-hydroxyethylureaand 1-nitroso-1-hydroxyethyl-3-ethyl- urea was that the formerinduced only neoplastic nodules in the,liver of 30% of the rats,while the latter induced hepatocellular carcinomas in 55% ofthe rats; approximately half of the rats given either compoundhad brain neoplasms, which included astrocytomas, gliomas andoligoden drogliomas.     

Alpha-acetoxy derivatives of methyl-2-oxopropylnitrosamine: synthesis, hydrolysis rate and bacterial mutagenicity

N-Methyl-N-2-oxopropylnitrosamine (MOP) induces pancreatic tumors in hamsters. As models for the putative proximate carcinogenic alpha-hydroxy derivatives, we studied N-acetoxymethyl-N-2-oxopropylnitrosamine (AMOP) and N-methyl-N-(1-acetoxy-2-oxopropyl)nitrosamine (MAOP). AMOP was synthesized from aminoacetone by the method of Roller et al. [1975), Tetrahedron Lett., 25, 2065-2068) and MAOP was synthesized by acetoxylation of MOP with lead tetraacetate. The half-lives of AMOP, MAOP and acetoxymethylmethylnitrosamine (ADMN) in aqueous buffer decreased as the pH rose from 5 to 9, with values at pH 5 of 2.8 X 10(4) min for AMOP, 3.2 X 10(3) min for ADMN, and 23 min for MAOP. Mutagenicity was examined in Salmonella typhimurium TA1535, using a pre-incubation at pH 5 without microsomal activation. The mutagenic potency, expressed as revertants/mumole, was 56 for AMOP, 150 for ADMN, and 4.5 X 10(4) for MAOP. Hence, hydrolysis rates at pH 5 were probably important in determining the relative mutagenicity.     

Synthesis of a new nor-aza-steroidal ester of p-N,N-bis-(2-chloroethyl)aminophenylbutyric acid and in vitro study of its mutagenicity and clastogenicity

A new nor-aza-steroidal ester of chlorambucil has been synthesized. The study of the mitotic index in CHO and HeLa cells treated with this compound showed that it may be a cytostatic drug. It was also found that treatment of CHO cells with a dose as low as 5 micrograms/ml induces a large number of sister chromatid exchanges. A great number of abnormal metaphases has been observed when CHO cells were treated with the compound at a dose of 25 micrograms/ml. When the compound was tested in the Ames/Salmonella microsome assay, it was found to be mutagenic in strains TA100 and TA1535, both with and without metabolic activation.     

Synthesis, mutagenicity, binding to pBR 322 DNA and antitumour activity of platinum(II) complexes with ethambutol

Platinum complexes with N,N'-bis(1-hydroxybut-2-yl)ethylenediamine, [PtCl2(ethambutol)] were prepared and the biological activity of three isomers [with (-), (+) and (+/-) ethambutol, respectively] investigated. All species interact with the Bam HI and Ava I recognition sequences showing a binding preference for GC rich sequences of DNA. The complex which showed the greatest interaction with adjacent guanines, [PtCl2[+/-)ethambutol)] was also found to be the most mutagenic of the three. On the other hand, only [PtCl2[+)ethambutol)] had a considerable antitumour activity against both P388 leukaemia and Lewis lung carcinoma, and this was not correlated either with restriction enzyme blocking activity or with mutagenicity.     

Tumor-initiating activity, mutagenicity, and metabolism of methylated anthracenes

Specific methylated derivatives of anthracene are mutagenicin S. typhimurium and have tumor-initiating activity on mouseskin. In this study, the mutagenic activities of 1-, 2-, and9-methylanthracene, 2,9- and 9,10-dimethylanthracene, 2,9, -10-trimethylanthracene,2,3,9,10-tetramethylanthracene, and the photo-oxide of 9,10-dimethylanthracenewere determined in S. typhimurium TA98 and TA100. The relativetumor-initiating activities of these compounds were also evaluated.These bioassays indicate that increased mutagenic potency andtumor-initiating activity are associated with the presence ofa methyl substituent at both the 9- and 10- position of anthracene.Metabolism studies suggest that the biological activity of specificmethylated anthracenes may be related to the formation of asimple epoxide adjacent to a peri-methyl substituent.     

Synthesis and mutagenicity of the diastereomeric fjord-region 11,12-dihydrodiol 13,14-epoxides of dibenzo [a,l]pyrene

Extensive tumongenicity studies in rodents revealed that dibenzo[a,l]pyrene(DB[a,l]P) is the most potent carcinogen among all polycyclicaromatic hydrocarbons (PAHs) tested so far. The structure ofthe genotoxic metabolite(s) responsible for this exceptionalcarcinogenicity is. unknown. The fjord-region syn- and anti-DB[a,l]P-11,12-dihydrodiol 13,14-epoxides (syn- and anti-DB[a,l]PDE)were synthesized to clarify their role as possible ultimatemutagenic and carcinogenic metabolites of DB [a,l]P.9-Formyl-11,12-dime-thoxybenzo [g] chrysense was prepared from 9-phenanthryl- aceticacid by a photochemical route. After reaction of the aldehydewith trimethylsulfonium iodide to generate an oxiranyl side-chain,treatment with boron trifluoride produced the key intermediate11,12-dimethoxy.DB[a,l]P in 14% overall yield. From 11,12-dimethoxy-DB[a,l]Pthe syn- and anti-DB[a,l]PDE were stereoselectively preparedvia the trans-11,12-dihydrodiol. The mutagenicity of the synand anti-DB[a,l]PDE was examined in four his^– strainsof Salmonella typhimurium and in Chinese hamster V79 cells.In all five test systems, the new dihydrodiolepoxides were morepotent than any of the previously investigated dihydrodlolepoxides.The specific mutagemcity observed with anti-DB[a,l]PDE in strainTA104 exhibited the highest value ever found with any compoundin any his strains of S.typhimurium. The same appears to hetrue for the activity observed with this compound in V79 cells.In all five systems, syn-DB[a,l]PDE was only moderately lessactive than its anti-dlastereomer (     

Effect of methyl substitution on mutagenicity of 2-aminodipyrido

The mutagenicities of 2-aminodipyrido [1,2-a:3',2'-d] imidazole (Glu-P-2, a mutagen obtained from pyrolysate of glutamic acid) and six methyl substituted derivatives of Glu-P-2 were tested with Salmonella typhimurium TA98 and TA100 with S-9 mix. Glu-P-1 (6-Me-Glu-P-2) was the strongest mutagen to TA98 and TA100. 3-Me-Glu-P-2 was weakest. The presence of a methyl group, and its position were shown to affect the mutagenicity significantly.     

丙烯酸-2-乙基己酯的遗传毒性研究

目的： 检测丙烯酸-2-乙基己酯的遗传毒性,为其安全性评价提供资料。方法：采用鼠伤寒沙门菌回复突变试验 (Ames试验),小鼠骨髓嗜多染红细胞微核试验和小鼠淋巴瘤细胞TK基因突变试验检测丙烯酸-2-乙基己酯的遗传毒性。Ames试验设每皿0.08、0.4、2.0、10.0和50.0 μL剂量组,另设空白、溶剂、阳性对照;微核试验设350、700和1 400 mg/kg剂量组,另设溶剂、阳性对照;TK基因突变试验设12.5、25、50和100 μmol/L剂量组,另设溶剂、阳性对照。 结果：Ames试验中,加或不加S9的情况下,丙烯酸-2-乙基己酯对TA97、TA98、TA100和TA102菌株均显示致突变作用。微核试验中,雌雄鼠微核率均呈现剂量-反应关系;雄鼠高剂量组微核率与对照组比较差异具有统计学意义(P<0.05);雌鼠各剂量组微核率与溶剂对照组比较差异无统计学意义(P>0.05)。TK基因突变试验中高剂量组突变频率与对照组比较,差异具统计学意义(P<0.05)。结论：在本实验条件下,Ames试验和TK基因突变试验均显示阳性结果,微核试验显示可疑阳性。丙烯酸-2-乙基己酯遗传毒性需结合其他检测系统综合评定。     

Effects of secondary biliary acids on the mutagenicity of N-methyl-N'-nitro-N-nitrosoguanidine, 2-acetylaminofluorene and 2-nitrofluorene towards Salmonella typhimurium strains

The two secondary biliary acids (lithocholic and deoxycholicacids)were co-mutagenic when they were each co-incubated withdimethylhydrazine in the presence of Salmonella typhimuriumTA 100. These observations were extended to other toxic chemicals,acting as direct (N-methyl-N'nitro-N-nitrosoguanidine and 2-nitrofluorene)or indirect (2-acetyl-aminofluorene)mutagens. Lithocholic anddeoxycholic acids show a similar behavior towards genotoxicmolecules. Nevertheless two differences must be noted. Lithocholicacid was the stronger co-mutagen. When lithocholic acid inhibitedmutagenic activity of 2-nitrofluorene, deoxycholic acid didnot modify it. Interactions between the two secondary bile acidsoccurred so that the co-mutagenic activity of the mixture ofthese two bile acids depended on the ratio of their concentrations.Besides their cancer-promoting effect, biliaryacids also couldhave co-initiating effect that could depend upon the ratio oftheir concentration in the intestine. Calculating the ratioof fecal concentrations of deoxycholic/lithocholic acids thuscould be a more sensitive index for cancer risk than simplymeasuring the fecal concentration of the two biliary acids takenseparately.     

Synthesis and mutagenicity of some cyclopenta[c]phenanthrenes and indeno[c]phenanthrenes

An efficient two-step synthesis of 8(H)-9,10,11,12-tetrahydrodicyclopenta[a,c]phenanthren-7-one,based on the high pressure Diels-Alder cycloaddition of 4-acetoxy-2-cyclopenten-1-onewith1-(1-naphthyl)cyclopentene and a subsequent dehydrogenation-aromatizationreaction, is reported. Further, the synthesis of two cyclopenta[c]-phenanthrenesand indeno[c] phenanthrenes is described. Structural analysisof the new products by ¹H and ¹³C NMR spectroscopy is presented.The mutagenic activity of the compounds in Salmonella typhimuriumwas estimated by Ames' test. Three compounds were shown to bemutagenic for the strain TA 100. The mutagenic activities exhibitedby cyclopenta[c]phenanthrenes are compared with those shownby the related cyclopenta[a]phenanthrenes and then discussedwith respect to the effect of the cyclopentane ring facing thebay region. Indeno[c]phenanthrenes are mostly inactive. Theeffect of benzoannulation on the mutagenic activities of cyclopenta[c]phenanthrenesis discussed.     

Persistence of DNA single-strand breaks and other tests as indicators of the liver carcinogenicity of 1-nitroso-5,6-dihydrouracil and the noncarcinogenicity of 1-nitroso-5,6-dihydrothymine

The cyclic nitrosourea 1-nitroso-5,6-dihydrothymine [(NDHT) 1-nitrosodihydrothymine] was not significantly carcinogenic when it was administered for 1 year in drinking water (206 mg/liter) to MRC-Wistar rats. In acute toxicity tests, ip injection of saline solutions of 1-nitroso-5,6-dihydrouracil [(NDHU) CAS: 16813-36-8; 1-nitrosohydrouracil], a strong liver carcinogen in rats, produced only mild liver toxicity but marked focal degeneration of myocardial fibers. NDHU injected ip in water solution produced subcapsular liver damage. NDHU, but not NDHT, induced unscheduled DNA synthesis in hepatocyte primary cultures. NDHU, NDHT, and methylnitrosourea [(MNU) CAS: 684-93-5; N-methyl-N-nitrosourea], a liver carcinogen only under special conditions, were tested for their ability, when injected ip into rats, to produce liver DNA damage measured as strand breaks by alkaline sucrose gradient centrifugation. The three nitrosoureas produced similar maximum DNA damage of 2.2-3.2 strand breaks/10(8) daltons. Eighty percent of the damage due to NDHU persisted for 7 days, and the damage at that time was significantly greater than that produced by NDHT and MNU. The varying persistence of liver DNA damage may explain why NDHU, but not NDHT, is a liver carcinogen.