Inhibition of the mutagenic effects of N-methyl-N'-nitro-N-nitrosoguanidine and 9-aminoacridine by indenopyridines in the Salmonella typhimurium tester strain 1537 and E. coli.

The goal of the present research was to determine the protective potential of five newly synthesized indenopyridine derivatives against N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and 9-aminoacridine (9-AA) induced mutagenesis. MNNG sensitive Escherichia coli WP2uvrA and 9-AA sensitive Salmonella typhimurium TA1537 were chosen as the bacterial tester strains. All of the test compounds showed significant antimutagenic activity at various tested concentrations. The inhibition rates ranged from 25.6% (Compound 2 - 1 mM/plate) to 68.2% (Compound 1 - 2.5 mM/plate) for MNNG and from 25.7% (Compound 4 - 1 mM/plate) to 76.1% (Compound 3 - 2.5 mM/plate) for 9-AA genotoxicity. Moreover, the mutagenicity of the test compounds was investigated by using the same strains. None of the test compounds has mutagenic properties on the bacterial strains at the highest concentration of 2.5 mM. Thus, the findings of the present study give valuable clues to develop new strategies for chemical prevention from MNNG and 9-AA genotoxicity by using synthetic indenopyridine derivatives.

Genotoxic and antigenotoxic assessment of four newly synthesized dihydropyridine derivatives.

The current study aims to determine the genotoxic and antigenotoxic potential of four newly synthesized dihydropyridine derivatives using Escherichia coli WP2 and Ames/Salmonella bacterial reversion assay systems. The bacterial mutant tester strains, E. coli WP2uvrA with a point mutation and Salmonella typhimurium TA1537 with a frameshift mutation, were used to determine genotoxic potentials of the test compounds. To determine antigenotoxic potentials of the test compounds, the same strains were also used together with positive mutagens N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) for E. coli WP2uvrA and 9-aminoacridine (9-AA) for S. typhimurium TA1537. According to the results, neither of the test compounds showed significant genotoxic activity on both tester strains at the tested concentrations. However, except compound 4, all the test compounds showed significant antigenotoxic activity on MNNG- or/and 9-AA-induced mutations. The inhibition rates of mutagenesis ranged from 27.0% (compound 2: 2.5 mM/plate) to 65.0% (compound 2: 0.5 mM/plate) for MNNG and from 30.6% (compound 2: 2 mM/plate) to 58.5% (compound 1: 1 mM/plate) for 9-AA genotoxicity. According to these results, it is concluded that all the test compounds do not have a mutagenic potential on the bacterial strains at the tested concentrations, and some of them have antigenotoxic potentials against MNNG- and 9-AA-induced mutagenesis.

Antigenotoxic properties of two newly synthesized ß-aminoketones against N-methyl-N'-nitro-N-nitrosoguanidine and 9-aminoacridine-induced mutagenesis.

The aim of this study was to determine the antigenotoxic potential of two newly synthesized ß-aminoketones against N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and 9-aminoacridine (9-AA)-induced mutagenesis. The mutant bacterial tester strains were MNNG-sensitive Escherichia coli WP2 uvrA and 9-AA-sensitive Salmonella typhimurium TA1537. Both test compounds showed significant antimutagenic activity at various tested concentrations. The inhibition rates ranged from 29.5% (compound 1: 2 mM/plate) to 47.5% (compound 2: 1.5 mM/plate) for MNNG and from 25.0% (compound 2: 1 mM/plate) to 52.1% (compound 2: 2.5 mM/plate) for 9-AA genotoxicity. Moreover, the mutagenicity of the test compounds was investigated by using the same strains. Neither test compound has mutagenic properties on the bacterial strains at the tested concentrations. Thus, the findings of the present study give valuable information about chemical prevention from MNNG and 9-AA genotoxicity by using synthetic ß-aminoketones.

Protective effects of methanol extracts from Cladonia rangiformis and Umbilicaria vellea against known mutagens sodium azide and 9-aminoacridine.

Lichens and their various extracts have been occasionally used in the treatment of many diseases. Cladonia rangiformis and Umbilicaria vellea are two important species of these lichens and they have several biological activities. In the present study, methanol extracts of these lichens, which are grown in the Eastern Anatolia Region of Turkey, were isolated, and their mutagenic and antimutagenic properties were investigated by using AMES-Salmonella and Zea mays Root Tip Mitotic Index mutagenicity and antimutagenicity assay systems. Known mutagens sodium azide (NaN(3)) and 9-Aminoacridine (9-AA) were used to determine antimutagenic properties of methanol extracts. The results showed that all methanol extracts, investigated in the present study, can be considered genotoxically safe because they do not have mutagenic activity at the tested concentrations. Besides, all of them have antimutagenic activity against 9-AA known as a model intercalator agent in the AMES-Salmonella test system. The inhibition rates obtained from the antimutagenicity assays ranged from 37.07% (C. rangiformis-5 µg/plate) to 54.39% (C. rangiformis-5 µg/plate). Furthermore, all the methanol extracts have significant antimutagenic activity against NaN(3) mutagenicity in Z. mays Root Tip Mitotic Index assay system. These activities are valuable towards an extension of the employ of these drugs as new phytotherapeutic or preservative ingredients.

Synthesis and evaluation of 9-aminoacridines derived from benzyne click chemistry.

A small set of 9-aminoacridine-3- and 4-carboxamides were synthesized efficiently using the benzyne/azide click chemistry. The products bind to duplex DNA but have different antitumour activity in the HL60 cell line.

Transformation of MCF-10A cells by random mutagenesis with frameshift mutagen ICR191: a model for identifying candidate breast-tumor suppressors.

BACKGROUND: Widely accepted somatic mutation theory of carcinogenesis states that mutations in oncogenes and tumor suppressor genes in genomes of somatic cells is the cause of neoplastic transformation. Identifying frequent mutations in cancer cells suggests the involvement of mutant genes in carcinogenesis. RESULTS: To develop an in vitro model for the analysis of genetic alterations associated with breast carcinogenesis, we used random mutagenesis and selection of human non-tumorigenic immortalized breast epithelial cells MCF-10A in tissue-culture conditions that mimic tumor environment. Random mutations were generated in MCF-10A cells by cultivating them in a tissue-culture medium containing the frameshift-inducing agent ICR191. The first selective condition we used to transform MCF1-10A cells was cultivation in a medium containing mutagen at a concentration that allowed cell replication despite p53 protein accumulation induced by mutagen treatment. The second step of selection was either cell cultivation in a medium with reduced growth-factor supply or in a medium that mimics a hypoxia condition or growing in soft agar. Using mutagenesis and selection, we have generated several independently derived cultures with various degrees of transformation. Gene Identification by Nonsense-mediated mRNA decay Inhibition (GINI) analysis has identified the ICR191-induced frameshift mutations in the TP53, smoothelin, Ras association (RalGDS/AF-6) domain family 6 (RASSF6) and other genes in the transformed MCF-10A cells. The TP53 gene mutations resulting in the loss of protein expression had been found in all independently transformed MCF-10A cultures, which form large progressively growing tumors with sustained angiogenesis in nude mice. CONCLUSION: Identifying genes containing bi-allelic ICR191-induced frameshift mutations in the transformed MCF-10A cells generated by random mutagenesis and selection indicates putative breast-tumor suppressors. This can provide a model for studying the role of mutant genes in breast carcinogenesis.

Attenuation of acridine mutagen ICR-191--DNA interactions and DNA damage by the mutagen interceptor chlorophyllin.

We have investigated the ability of chlorophyllin (CHL) to interact with acridine mutagen ICR-191 (2-methoxy-6-chloro-9-(3-(2-chloroethyl)aminopropylamino)acridine) and also its ability to decrease binding of ICR-191 to DNA in a simple three-component competition system: CHL-ICR-DNA. Our data indicate a strong association of ICR-191 with CHL, stronger even than the association of ICR-191 with DNA. Calculations based on the measured affinity data show that a two- to three-fold excess of CHL reduces by about two-fold the concentration of the mutagen-DNA complex. We also exposed human leukemic HL-60 cells to ICR-191 in the absence and presence of CHL and measured the mutagen-induced DNA damage. The extent of DNA damage was assessed by analysis of histone H2AX phosphorylation. While ICR-191 induced significant increase in expression of phosphorylated H2AX (gammaH2AX), particularly in DNA replicating cells, this increase was totally abolished in the cells treated with ICR-191 in the presence of CHL.

Isolation and characterization of novel phenotype CHO cell mutants defective in peroxisome assembly, using ICR191 as a potent mutagenic agent.

To elucidate molecular and cellular mechanisms of peroxisome biogenesis, we have isolated Chinese hamster ovary (CHO) cell mutants defective in peroxisome biogenesis by making use of enhanced green fluorescent protein (EGFP) and a frameshift-inducing mutagen ICR191. CHO-TKa cells stably expressing Pex2p were transformed with a cDNA encoding EGFP fused with peroxisomal targeting signal type 2 (PTS2-EGFP), termed Tka/EG2. TKa/EG2 cells were mutagenized with ICR191 and cultured in the presence of P9OH (9-(1'-pyrene) nonanol) followed by an exposure to UV. P9OH/UV-resistant and morphologically peroxisome-deficient mutant cells were isolated by directly observing cytosolic localization of EGFP, without cell staining. By a combination of cell-fusion and PEX transfection, we determined complementation groups (CGs) of 16 cell mutants isolated here. The mutants were classified into five CGs, including pex2, pex3, pex5, pex6, and pex7 cell mutants. In contrast to typical pex6 mutants with the impaired import of both PTS1- and PTS2-proteins, two clones, ZPEG236 and ZPEG244, showed a distinct, novel phenotype where PTS1-protein import was normal despite the abrogated PTS2 import. Dysfunction of Pex3p in pex3 ZPEG 238 was due to one base (G) insertion in the codon for Asn7 resulting in a frameshift, thereby inducing a distinct 31 amino-acid sequence and a termination. pex2 ZPEG239 showed a mutation in codon GAG for Glu(201) to a nonsense mutation, TAG. Thus, the method developed here using ICR191 could be useful for isolation of further novel cell mutants impaired in peroxisome biogenesis.

Disruption of DNA Damage-Response by Propyl Gallate and 9-Aminoacridine.

The DNA-damage response (DDR) protects the genome from various types of endogenous and exogenous DNA damage, and can itself be a target of certain chemicals that give rise to chromosomal aberrations. Here, we developed a screening method to detect inhibition of Mediator of DNA damage Checkpoint 1 (MDC1) foci formation (the Enhanced Green Fluorescent Protein (EGFP)-MDC1 foci formation-inhibition assay) using EGFP-MDC1-expressing human cells. The assay identified propyl gallate (PG) and 9-aminoacridine (9-AA) as inhibitors of camptothecin (CPT)-induced MDC1 foci formation. We demonstrated that the inhibition of CPT-induced MDC1 foci formation by PG was caused by the direct suppression of histone H2AX phosphorylation at Ser139 (γH2AX), which is required for MDC1 foci formation, by quantifying γH2AX in cells and in vitro 9-AA also directly suppressed H2AX Ser139-phosphorylation in vitro but the concentration was much higher than that required to suppress CPT-induced MDC1 foci formation in cells. Consistent with these findings, PG and 9-AA both suppressed CPT-induced G2/M cell-cycle arrest and increased the number of abnormal nuclei. Our results suggest that early DDR-inhibitory effects of PG and 9-AA contribute to their chromosome-damaging potential, and that the EGFP-MDC1 foci formation-inhibition assay is useful for detection of and screening for H2AX Ser139-phosphorylation-inhibitory effects of chemicals.

Specificity of base substitutions induced by the acridine mutagen ICR-191: mispairing by guanine N7 adducts as a mutagenic mechanism.

As the most nucleophilic site in DNA, the guanine N7 atom is a major site of adduction by a large number of alkylating mutagens and carcinogens. Aflatoxin B1, a powerful mutagen, is believed to act through its reaction with this DNA site. On the basis of the specificity of base substitutions induced by various adduct forms of aflatoxin, we have proposed that bulky guanine N7 adducts elicit base substitutions by two mechanisms. The first mechanism is similar to that observed for a number of bulky noninstructive lesions, whereas the second mechanism invokes mispairing between N7-adducted guanine and thymine. A prediction of the mispairing hypothesis is that diverse bulky guanine N7 adducts (regardless of structural similarities with the aflatoxins) should induce predominantly G-to-A transitions. Accordingly, we have recently observed that base substitutions induced by the acridine half-mustard ICR-191 in the M13 double-stranded DNA transfection system are predominantly G:C-to-A:T transitions. Here, by transfecting ICR-191-treated M13 AB28 single-stranded DNA into Escherichia coli, we show that base substitutions are predominantly targeted to guanines. Since the N7-adducted-guanine:thymine mispairing is proposed to require N1 deprotonation promoted by the primary N7 lesion, guanine imidazole ring-opening should abolish this mispairing property, and thereby alter the specificity of mutagenesis. Here, we show that the incubation of ICR-191-treated RF DNA at pH 10.5 results in a significant reversal of the specificity of G:C-targeted substitutions such that G-to-T transversions predominated over G-to-A transitions.(ABSTRACT TRUNCATED AT 250 WORDS)

Induced mutation spectra at the upp locus in Salmonella typhimurium: response of the target gene in the FU assay to mechanistically different mutagens.

A novel forward mutation assay has been developed in Salmonella typhimurium based on resistance to 5-fluorouracil (FU). The mutational target in the FU assay was determined to be the uracil phosphoribosyl transferase (upp) gene. To validate the upp gene as a suitable target for monitoring a variety of induced mutations, the mutational specificity was determined for five mechanistically different mutagens. The mutagens included a polycyclic hydrocarbon (benzo[a]pyrene, B[a]P), SN1 and SN2 alkylating agents (N-nitroso-N-methylurea, MNU, and methyl methanesulfonate, MMS, respectively), a frameshift mutagen (ICR-191), and an oxidative-damaging agent (hydrogen peroxide, H2O2). Induced mutation frequencies were measured in the presence and absence of the plasmid pKM101 (strain FU100 and FU1535, respectively). pKM101 renders FU100 more susceptible to induced mutation by providing error-prone replicative bypass of DNA adducts. B[a]P, MMS, and H2O2 failed to induce the mutant frequency in FU1535, demonstrating the dependence of pKM101 on induced mutations with these agents. ICR-191 and MNU were not dependent on pKM101, and did significantly induce mutations in FU1535. In contrast to FU1535, all agents significantly induced mutations in FU100. Approximately 60 independent mutants were sequenced for each agent that significantly induced the mutant frequency above background. The resulting mutational spectra illustrated predictable molecular fingerprints based on known mutagenic mechanisms for each agent. The predominant mutations observed were G:C to T:A transversions for B[a]P, A:T to T:A and G:C to T:A transversions for MMS, G:C to T:A transversions and A:T frameshifts for H2O2, G:C frameshifts for ICR-191, and G:C to A:T transitions for MNU. It can be concluded that the upp gene in the FU assay is a sensitive and suitable target to monitor a variety of induced mutations in Salmonella.

Frameshift mutations induced by the acridine mustard ICR-191 in embryos and in the adult gill and hepatopancreas of rpsL transgenic zebrafish.

To determine whether frameshift mutations can be detected in rpsL transgenic zebrafish (Brachydanio rerio), embryos, and adult fish were treated with 6-chloro-9-[3-(2-chloroethylamino)-propylamino]-2-methoxyacridine (ICR-191). Embryos exposed to 0, 10, or 20 microM ICR-191 in a water bath for 18 h exhibited induced mutant frequencies (MFs) of 14 x 10(-5), 16 x 10(-5), and 25 x 10(-5), respectively. Only embryos exposed to 20 microM ICR-191 showed a significant increase in MF. The mutational spectra differed between the control and ICR-191-treated groups and single G:C pair insertions, which are a marked characteristic of ICR-191 mutagenesis, were observed in both 10 and 20 microM-treated embryos. In adult fish treated with 1 microM ICR-191 in a water bath for 18 h, a significant increase in MFs was observed in both gill (12 x 10(-5) and 44 x 10(-5) in control and treated fish, respectively), and hepatopancreas (5 x 10(-5) and 29 x 10(-5), respectively) 2 weeks after exposure. Sequence analysis showed that 58% of mutations in gill and 94% of mutations in hepatopancreas were single G:C pair insertions, which is typical of mutations induced by ICR-191. Additionally, these mutations occurred predominantly at a single site (CC sequence at bps 140-141) in the rpsL gene. Three weeks after exposure, however, the increased MFs and prominent mutational spectra of ICR-treated fish were undetectable. These findings suggest that using our protocols the rpsL transgenic zebrafish mutation assay is more effective for adult fish than for embryos, but that frameshift mutations can be detected in both embryos and adults at appropriate sampling times after treatment with ICR-191.

Re-analysis results using medians of the data from the JaCVAM-organized international validation study of the in vivo rat alkaline comet assay.

The data from the JaCVAM-organized international validation study of the in vivo rat alkaline comet assay were reported and analyzed statistically using the simple means of % tail DNA. However, OECD test guideline TG 489 recommends use of the median for data analysis due to the hierarchical nature of the data. Comparison between the simple mean approach and the median based approach for positive/negative/equivocal chemical calls was conducted using the % tail DNA data for the 40 chemicals tested in the JaCVAM-organized international validation study of the in vivo rat alkaline comet assay, using liver and stomach as target organs. In the liver, two genotoxic chemicals, o-anisidine and 9-aminoacridine hydrochloride monohydrate, were positive using the median based approach but negative using the simple mean approach, and two genotoxic chemicals, 2-acetylaminofluorene and busulfan were equivocal using the median based approach but negative using the simple mean approach. In contrast, cadmium chloride (genotoxic carcinogen) was equivocal in both organs using the median based approach, while positive and equivocal in liver and stomach, respectively, using the simple mean approach. Two data sets of sodium arsenite showed equivocal and negative results for liver using the median based approach, although both data sets were equivocal using the simple mean approach. Overall, there are no large differences in terms of the genotoxic call between both approaches. However, the median based approach recommended in OECD TG 489 has an advantage toward higher precision within the groups treated with a test chemical, whereas the approach might show the lower values for the effect.

In vivo comet assay of acrylonitrile, 9-aminoacridine hydrochloride monohydrate and ethanol in rats.

As part of the Japanese Center for the Validation of Alternative Methods (JaCVAM)-initiative international validation study of the in vivo rat alkaline comet assay, we examined the ability of acrylonitrile, 9-aminoacridine hydrochloride monohydrate (9-AA), and ethanol to induce DNA damage in the liver and glandular stomach of male rats. Acrylonitrile is a genotoxic carcinogen, 9-AA is a genotoxic non-carcinogen, and ethanol is a non-genotoxic carcinogen. Positive results were obtained in the liver cells of male rats treated with known genotoxic compounds, acrylonitrile and 9-AA.

Nitroaryl compounds as potential fluorescent probes for hypoxia. II. Identification and properties of reductive metabolites.

Nitroakridin 3582 (NA) and a nitronaphthalimide (DM113), which fluoresce only upon reduction, have been studied by HPLC. V79-379A cells incubated with NA under 20% or 2% O2 and N2 gave increasing amounts of the fluorescent amine with an hypoxic:oxic differential of 160. Measurement of the uptake of NA showed that it was concentrated within the cell by over 1000-fold. Studies in 3 different cell lines of reduction under hypoxia showed a 7-fold range in amine production. DM113 yields more than one fluorescent product, which show different absorption and fluorescence spectra. Chemical reduction of NA or DM113 using a variety of methods gave, depending on conditions, amine and/or (what was presumed to be) hydroxylamine; the latter was non-fluorescent. In vivo, NA is toxic at greater than 0.19 mumol g-1. At this dose much of the drug is found in the liver and kidneys. Plasma levels at 30 minutes are only 2 microM while tumor concentrations are 10 microM compared to 600 microM in the liver. However, the half life is greater than 1 hr and amine was detectable in these tumors.

Frameshift mutations induced by three classes of acridines in the lacZ reversion assay in Escherichia coli: potency of responses and relationship to slipped mispairing models.

The frameshift mutagenicity of 9-aminoacridine (9AA) was compared with that of quinacrine, the acridine mustards ICR-191 and quinacrine mustard (QM), and the nitroacridine Entozon in the lacZ reversion assay in Escherichia coli. As intercalating agents, 9AA and quinacrine cause mutations through noncovalent associations with DNA. Mustards and nitroacridines form covalent adducts in DNA and give rise to different spectra of mutations. Quinacrine and 9AA most effectively induced -1 frameshifts in a run of guanine residues, with 9AA being the more potent mutagen. They also induced +G frameshifts. The acridine mustard ICR-191 was a stronger mutagen than 9AA, owing largely to its potent induction of +G frameshifts. QM induced +G frameshifts more strongly than did its nonreactive counterpart quinacrine. The nitroacridine Entozon differed from the other acridines in being a potent inducer of -2 frameshifts, but it was less effective in inducing +/-1 frameshifts. Quinacrine, although a simple intercalator, induced all five kinds of frameshift mutations detected in the assay, as did the acridine mustards. Although +A and -A frameshifts were induced, adenine runs were less susceptible to acridine mutagenesis than guanine runs. The patterns of frameshift mutagenicity in the lacZ assay are similar to those in an assay based on the reversion of mutations in the tetracycline-resistance gene of the plasmid pBR322. The similarity suggests that the responses reflect the inherent bacterial mutagenicity of the compounds in the local sequence context and are not highly dependent on the broader sequence context. The results are interpreted with respect to slipped mispairing models of frameshift mutagenesis.

Mutational spectrum of ICR-191 at the hprt locus in human lymphoblastoid cells.

Human TK6 lymphoblasts were treated with the acridine derivative ICR-191, and mutants at the hprt locus were isolated. Mutant hprt cDNA was reverse-transcribed from mRNA, amplified by polymerase chain reaction (PCR), and sequenced. Additions of single G:C base pairs (+1 frameshift mutations) in repetitive G:C sequences were found in 82% (32/39) of the mutants. Sixteen of the +1 frameshifts analyzed were located in a single sequence of six consecutive guanine bases in exon 3. The remaining +1 frameshifts occurred at six different GGG sequences (14 mutants) and a single GGGG sequence (2 mutants) in other hprt exons. The repetitive guanine sequences that underwent frameshift mutagenesis were located in both the transcribed and nontranscribed strands of hprt. No single base deletions (-1 frameshift mutations) were observed. Base substitutions were observed in 13% (5/39) of the clones analyzed and occurred at both G:C and A:T bases. Loss of exon 4 from the cDNA was also observed in 5% (2/39) of the mutants. Hprt mutants containing seven consecutive guanines (produced from a +1 frameshift in a GGGGGG sequence) were treated with ICR-191 and wild-type revertants selected in CHAT medium. Revertants were recovered at a frequency of approximately 10(-7) and contained the wild-type sequence (GGGGGG) in all clones analyzed. The observed frequency of ICR-191-induced-1 frameshift reversion in the GGGGGGG sequence was approximately 500-fold lower than the estimated frequency of +1 frameshifts observed in the wild-type GGGGGG sequence following the same ICR-191 treatment. These results suggest that ICR-191 produces predominantly +1 frameshift mutations at the hprt locus in human cells.

Preclinical pharmacologic investigations on a new group of acridine derivatives with oncostatic activity. I. Acute and subchronic action.

The influence of l-nitro-9-/2-dihydroxyethylamino-ethylamino-/-acridine (C-835) and l-nitro-9/3-izopropylaminopropylamino/-acridine (C-846) on the basic functions of animal organism in acute and subchronic experiments was studied. Both compounds displayed distinct all-biologic activity. Their toxicity (LD50) in mice was contained in milligramme interval (i.v.), while LD50 administered into the stomach was several dozen times higher. Even administration in protective phosphate buffer, which provides the most neutral pH, produced quite strong local irritant action. The range between LD50 and LD10 was relatively small, which proved relatively small tolerance factor. Both preparations injected intravenously in the doses starting from several mg/kg had hypotensic influence due to their affinity with the myocardium, the parasymphatetic system and the vascular system. The preparations acted spasmolytically on intestine muscles both in vivo and in vitro in a number of animal species. Central action showed clear stimulating component in a behavioric test. The effect of the compounds in interaction to hypnotic and convulsant drugs was equivocal and dependent on the dose and the reference-preparation. The examined compounds did not influence reproduction processes and did not display the teratogenic action. They impaired only the speed of growth of the newborns. The effect of both examined compounds was qualitatively and quantitatively similar.

Preclinical pharmacologic investigations on a new group of acridine derivatives with oncostatic activity. II. Chronic action.

Influence of 1-nitro-9-(2-dihydroxyethylaminoethylamino)-acridines (C-835) and 1-nitro-9-(3-isopropylamino-propylamino)-acridines (C-846) on animal organs and their basic functions was studied in the chronic action. Rabbits responded to the drugs with considerable tolerance which was less manifested in rats. Slight toxic effect was observed only after maximal doses with which the animals survived 3-month exposure. Enzyme studies and creatinine clearance test did not show any clear-cut impairment of hepatic and renal function. Neither did the preparations influence the red and white blood system, nor the blood clotting time. Microscopic examinations showed their slight (initially) morphologic changes which later passed into degenerative changes (liver, kidney, myocardium) as well as typical of oncostatics, changes in intestinal villi, nuclei and lymphatic system. Also desquamation of intestinal epithelium, hypertrophy of their lamina propria, impairment of spermato- and spermiogenesis as well as atrophy of lymphatic germinal centers and decreased number of small lymphocytes in lymph nodes and spleen were noted. Deviation between the results of function and morphologic tests were discussed. Microscopic changes observed after treatment of the tested acridine derivatives were considered too small to damage the particular organs function.

DNA damage in HeLa s3 cells by an antitumor drug Ledakrin and other antitumor 1-nitro-9-aminoacridines.

Ledakrin and seven other antitumor and cytotoxic derivatives of 1-nitro-9-aminoacridine were shown to induce DNA-single strand breaks in HeLa S3 cells as found by alkaline sucrose gradient centrifugation. The induced DNA damage is of non-random character. Some of Ledakrin -induced DNA breaks are probably generated by endonucleolytic cleavage in the course of repair processes as indicated by experiments with Novobiocin, an antibiotic preventing the incision step of DNA repair. Other Ledakrin -induced DNA breaks observed on alkaline sucrose gradients may arise from alkali-labile sites in DNA. Most of such sites seem to be converted to breaks after brief exposure to alkali. The extent of DNA damage by 1-nitro-9-aminoacridines was found to be correlated with cytotoxic activities of these compounds against HeLa S3 cells. Furthermore, Ledakrin and other derivatives seem to induce DNA-repair synthesis in HeLa S3 cells as judged by the stimulation of hydroxyurea (HU)-resistant incorporation of [3H] thymidine into DNA. The agents studied differ in their concentrations required to produce a considerable stimulation of DNA repair, whereas the maximal level of this effect is similar for all the derivatives assayed. The former values are correlated with cytotoxic activities of these compounds and seem to reflect the overall extent of DNA damage by 1-nitro-9-aminoacridines. Stimulation of DNA-repair synthesis is gradually shut off during prolonged incubation of the cells with Ledakrin or during postincubation of the cells in a drug-free medium. Such postincubation results also in the gradual accumulation of DNA-single strand breaks as observed by alkaline sucrose centrifugation. Hence, HeLa S3 cells are incapable of efficiently removing DNA damage by 1-nitro-9-aminoacridines, though the drug's action activates temporarily some repair mechanisms. The reported results suggest that overall DNA damage may contribute to the cytotoxic effects of 1-nitro-9-aminoacridines besides previously found ability of these agents to form interstrand DNA cross-links.