On the persistence of the radioprotective effect of chlorophyllin (CHLN) in somatic cells of Drosophila

By delaying the time of gamma irradiation of 72 h larvae, pretreated at 48 h with 5% chlorophyllin (CHLN), it was established that the overall inhibiting effect of CHLN in somatic cells of Drosophila, as measured in the wing spot test, persists for about 4 days or until the time of cessation of the proliferation of wing anlagen. In the same population of cells, some spot classes gave evidence of an inhibitory effect whereas others did not arguing against the suggestion that the radioprotective effect of CHLN is a consequence of an induced delay in development, shrinking of the potential radiation target and lowering the probability of induced events. Other observations of interest are described.     

Evidence that chlorophyllin (CHLN) may behave as an inhibitor or a promoter of radiation-induced genetic damage in somatic cells of Drosophila

Irradiation of 96 h old Drosophila following a 24 h pretreatment with 5% chlorophyllin (CHLN) was delayed 0–4 days. The antimutagenic effect of CHLN in somatic cells monitored by the wing spot test persisted for 3 days after completion of the pretreatment and appeared to terminate at a time corresponding to the cessation of mitotic divisions of wing anlagen cells. Within the same population of cells, CHLN demonstrated both an inhibitory effect as measured in mwh single spot classes, and contrarily, a promoting effect in the class of mwh/flr twin spots and to an extent in the class of large flr spots. The reason for the contrasting effects of CHLN remains to be determined.     

Evidence that chlorophyllin (CHLN) may behave as an inhibitor or a promoter of radiation-induced genetic damage in somatic cells of drosophila

Irradiation of 96h old Drosophila following a 24h pretreatment with 5% chlorophyllin (CHLN) was delayed 0-4 days. The antimutagenic effect of CHLN in somatic cells monitored by the wing spot test persisted for 3 days after completion of the pretreatment and appeared to terminate at a time corresponding to the cessation of mitotic divisions of wing anlagen cells. Within the same population of cells, CHLN demonstrated both an inhibitory effect as measured in mwh single spot classes, and contrarily, a promoting effect in the class of mwh/flr twin spots and to an extent in the class of large flr spots. The reason for the contrasting effects of CHLN remains to be determined.     

Evidence suggesting that chlorophyllin (CHLN) may act as an inhibitor or a promoter of genetic damage induced by chromium(VI) oxide (CrO3) in somatic cells of Drosophila

In Drosophila, 48h-old larvae were pretreated for 24h with chlorophyllin (CHLN) or sucrose and then treated with chromium(VI) oxide (CrO(3)) immediately following completion of the pretreatment period (0-day delay) or delayed 1, 2 or 3 days. The effects were scored in the wing spot test. After delays of 0 and 1 day, clear evidence of a protective effect of CHLN was found. Contrarily, after delays of 2 and 3 days, the results showed a reversal, i.e. CHLN-related events appeared more frequently than those in the sucrose control suggesting a promoting effect. It would appear prudent that CHLN be tested in a variety of situations in any given organism before decisions are reached regarding its inhibitor/promoter effects.     

Humic acids inhibit the formation but not the mutagenicity of N-methyl-N-nitrosourea

Humic acids in the form of potassium humate (KH), at concentrations exerting a strong inhibitory effect on the formation of N-methyl-N-nitrosourea (MNU) when present during the nitrosation of N-methylurea (MU) at pH 3, did not reduce the mutagenicity of preformed MNU in Tradescantia, clone 4430. The inhibitory effect of 20 mg/ml KH corresponds approximately to that of 3.75 mM (0.66 mg/ml) ascorbic acid towards the formation of MNU from the mixture of 7.5 mM MU + 7.5 mM NaNO2.     

Mutagenicity of cyanate, a decomposition product of MNU

Knox reported that the short-term effects of the carcinogen methylnitrosourea (MNU) were due to the formation of its decomposition product, the cyanate ion. He showed that cell survival and DNA synthesis decreased as the concentration of MNU and the cyanate ion (NCO-) increased in the medium. Further, the product of MNU decomposition comigrated with NCO- when added to his chromatographic test system. However, Knox did not study the mutagenicity of MNU or its breakdown products. We compared the mutagenicity of MNU and potassium cyanate (KNCO) in mammalian cells. Our results demonstrate that, although it is toxic to cells, KNCO does not induce ouabain-resistant mutants in cultured Chinese hamster cells (V79).     

On the persistence of the radioprotective effect of chlorophyllin (CHLN) in somatic cells of Drosophila

This research was designed to examine the presence of mutagenic/carcinogenic compounds in urban airborne particulates sampled with the inhalable PM-10 high volume sampler in two different streets of Brescia, a heavily industrialized town in northern Italy, using the Tradescantia/micronucleus test and a bacterial mutagenicity test (Kado test, a more sensitive version of the Ames test). In addition, the Tradescantia/micronucleus test was used for in situ monitoring of gaseous pollutants in other urban areas of Brescia and in two car tunnels, one with heavy car traffic in Perugia, a town in central Italy, and one in Brescia with moderate traffic. The Tradescantia-micronucleus test carried out on extracts of airborne particulates gave positive results only for the sample collected in the traffic-congested street where also higher bacterial mutagenicity was found. The in situ monitoring of the urban areas with the Tradescantia/micronucleus test always gave negative results. Monitoring carried out in the two car tunnels showed a significant increase in micronuclei frequency only in flowers exposed in the smaller and more polluted tunnel.     

Differential mutagenicity of streptozotocin analogs of the carbohydrate moiety

The mutagenicity of streptozotocin (SZN), 8 of its analogs and N-msthyl-N-nitrosourea (MNU) were compared in Salmonella typhimurium. SZN and its analogs carry MNU attached to the carbohydrate moiety at the C-2 position. The C-1 analogs tested were α- and β-methyl-SZN, α-ethyl-SZN, β-propyl-SZN, α- and β-butyl-SZN; in 2 analogs glucose was replaced by α- or β-inositol. When the ability of these compounds to revert the hisG46 auxotroph was compared, they fell into 4 groups which differed by about 10-fold in mutagenicity from one another. The most mutagenic was (i) SZN, followed by (ii) β-methyl-SZN; (iii) α-methyl-SZN, α-ethyl-SZN, β-propyl-SZN, α- and β-butyl-SZN; (iv) α and β-inositol-MNU. These results suggest that the presence of the glucose moiety is conducive to a high level of mutagenicity of SZN. Alterations of the glucose moiety by addition of larger alkyl groups, especially in the α position lead to decreased mutagenicity. The least mutagenic analogs are those in which the glucose moiety is replaced by inositols.The mutagenicity of SZN, β-methyl-SZN and of β-butyl-SZN was also compared in a mouse tissue-mediated assay. SZN was about 500-fold more mutagenic than its β-methyl analog, while the β-butyl analog was not mutagenic.Depletion of SZN and 4 of its analogs from the medium in presence of bacteria was determined spectrophotometrically. The more mutagenic compounds were depleted more rapidly but the quantitative differences in mutagenicity between these compounds could not be accounted for by depletion alone.     

Effects of sodium selenite and caffeine on mutagenesis induced by N-methyl-N-nitrosourea, N-methyl-N'-nitro-N-nitrosoguanidine and aflatoxin B1 in S. typhimurium.

Pre-treatment, co-treatment, and post-treatment procedures were comparatively used in order to assess the modulation of mutagenicity in S. typhimurium his- strains. Pre-treatment of bacteria with sodium selenite had no effect on sodium azide mutagenicity. Irrespective of the procedure used neither selenite nor caffeine had any influence on the S9-mediated mutagenicity of aflatoxin B1. In contrast, the mutagenicity of N-methyl-N-nitrosourea (MNU) and N-methyl-N'-nitro-N- nitrosoguanidine (MNNG) was variably affected, depending on the sequence of exposures of target bacterial cells to mutagens and modulators. In particular, pre-treatment of bacteria with either selenite or caffeine or their combination generally resulted in a potentiation of MNU and MNNG mutagenicity. However, co-incubation of these alkylating agents and test modulators with bacterial cells yielded an evident inhibition of mutagenicity, the methylxanthine being more effective in this case. Caffeine exhibited an an antimutagenic effect towards MNU also when assayed in a post-treatment procedure. Thus, in dependence on the test conditions, selenite and caffeine could act in the same mutagenicity assay as co-mutagens, antimutagens or agents without effect on mutagenesis. These opposite trends reflect the complexity of the mechanisms of action of both mutagens and modulators tested, and underscore the variable outcome of their interactions, also depending on topological and chronological factors. The data reported emphasize the need for a multiple methodological approach in studies investigating the modulation of mutagenicity.     

Aphidicolin allows a rapid and simple evaluation of DNA-repair synthesis in damaged human cells

The decrease in microbial mutagenicity of N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) and N-methyl-N-nitrosourea (MNU) was compared in an animal mediation with rats and in direct incubation with human as well as rat blood and blood components. The mutagenic activity was assayed by reverse mutation from streptomycin (SM) dependence to non-dependence in Escherichia coli, strain Sd-B (TC). The mutagenic response curves of both MNNG and MNU were approximately linear and parallel at non-cytotoxic concentrations. However, the mutagenic capabilities of MNNG were estimated to be 10-fold more potent than those of MNU. The mutagenic activity in blood and liver preparations from rats killed immediately after intravenous injection of MNNG, 50 mg/kg, was negative. Results with MNU, 100 mg/kg, were positive in both cases.For the detection of mutagenicity, blood was diluted 50 times for the final testing mixture (1 ml) to avoid bactericidal effects of the blood itself. When a larger amount of liver preparation was used in the tests, and diluted 8 times, mutagenic activity was still detected 15 min after injection of MNU, 80 mg/kg. Comparisons of the diminished rate of mutagenicity between MNNG and MNU during certain periods of incubation with blood indicated that MNNG was inactivated much more rapidly than MNU with both human and rat blood. Plasma showed a moderate inactivating effect on both MNNG and MNU. Red blood cells inactivated MNNG at a remarkably rapid rate similar to that of whole blood, but was less effective on MNU. In further experiments with red- cell components, the cell contents inactivated both MNNG and MNU at rates similar to those with red cells, but cell membrane had absolutely no effect in decreasing the mutagenicity in either MNNG or MNU.     

Chlorophyllin protects HEp-2 cells from nuclear fragmentation induced by poliovirus

AIMS: Chlorophyllin (CHLN) is a synthetic derivative of chlorophyll that possesses antimutagenic activity against several environmental contaminants. In the present study, CHLN was assayed for its capacity to prevent nuclear fragmentation (NF) in HEp-2 cells infected with poliovirus. METHODS AND RESULTS: CHLN was assayed at concentrations of 0.5 and 2.5 microg ml(-1), and NF was monitored using the comet assay and acridine orange staining. We demonstrated that CHLN reduced the percentage of NF in poliovirus-infected HEp-2 cells, when cells were treated with drug before infection or exposed continuously to drug. However, the highest degree of protection was achieved when the virus was exposed to CHLN before infection followed by protocol where infected cultures were continuously exposed to the drug after infection. CONCLUSIONS: It is suggested that CHLN primarily binds to the virus which inhibits cell penetration, thereby maintaining nuclear integrity. SIGNIFICANCE AND IMPACT OF THE STUDY: Considering that CHLN has several beneficial properties and no significant toxic effects in humans and animals, it would be an ideal candidate drug to test for antiviral activity.     

Two types of antimutagenic effects of gallic and tannic acids towards N-nitroso-compounds-induced mutagenicity in the amesSalmonella Assay

The frequency ofhis ⁺ revertants induced by N-methyl-N-nitrosourea (MNU) and N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) in the strain TA100 ofSalmonella typhimurium was decreased by gallic and tannic acid. In weak buffer solutions, the inhibition effects of gallic acid towards MNU and MNNG mutagenicity was caused primarily by a decrease of pH in the incubation mixtures. At adjusted pH (pH 5.0 and 6.5), the antimutagenic effects are largely the result of an interaction between MNU or MNNG with phenolic acids outside the cells.     

Role of O6-methylation in the initiation of GGTase-positive foci

The ability of seven methylating agents to form 7-methylguanine and O6-methylguanine was compared to their ability to initiate carcinogenesis as measured by the initiation of GGTase-positive foci. The seven methylating agents studied were methyl-N-nitroso-p-toluenesulfonamide (diazald), dimethylhydrazine (DMH), dimethylnitrosamine (DMN), dimethylsulfate (DMS), methyl methanesulfonate (MMS), methyl-N-nitro-N-nitrosoguanidine (MNNG) and methyl-N-nitrosourea (MNU). The DNA methylation and initiation of GGTase-positive foci was determined in partial hepatectomized rats. The formation of foci was promoted by 500 ppm sodium phenobarbital in the drinking water. While six of the seven compounds (DMH, DMN, DMS, MMS, MNNG and MNU) produced 7-methylguanine, only the four compounds (DMH, DMN, MNNG and MNU) that produced O6-methylguanine initiated GGTase-positive foci. The extent of O6-methylguanine produced by the methylating agents did not correspond with their potency to initiate GGTase-positive foci. Therefore, the initiation of GGTase-positive foci required the formation of O6-methylguanine. However, some sequential event altered the quantitative relationship of O6-methylguanine formation to the incidence of GGTase-positive foci.     

Role of the carbamoylation reaction in the biological activity of methyl nitrosourea

Study of the mutagenic action of methyl nitrosourea (MNU) on the CHO-AT3-2 Chinese hamster cell at 2 regimes of cell treatment (a short-term regime and prolonged 1-h treatment) revealed that increase in the duration of treatment enhanced both cell lethality and clastogenic and mutagenic effects at the TK locus and did not influence the mutation frequency at the OUAr locus. On the basis of kinetic considerations it can be concluded that the base-pair substitution-type mutants (e.g., OUAr) appear as a result of DNA alkylation and the mutants at loci with a wide spectrum of registered mutants (the TK locus) are related to a greater extent to the carbamoylating activity of MNU. This conclusion is confirmed by measurements of the effects of sequential treatment with MNU (7 min) and KNCO (1 h). A synergistic increase in lethality, clastogenicity and mutagenicity at the TK locus was found in experiments with the combined treatment of cells with ethyl methanesulfonate (EMS) and KNCO. Besides, pretreatment of cells with potassium cyanate and subsequent exposure to MNU, EMS and benzopyrene (BP) produced synergistic effects in all the tests: lethality, clastogenicity and mutation frequency at the OUAr and TK loci. Posttreatment of cells with KNCO also led to a synergistic increase in the effects of MNU, EMS and BP treatment in several tests, but not in the OUAr locus. The possible mechanism and levels of interactions between alkylation and carbamoylation and the possibility that potassium cyanate causes supramolecular lesions are discussed.     

Effects of chlorophyllin on replication of poliovirus and bovine herpesvirus in vitro

Aims:  Chlorophyllin (CHLN), a synthetic derivative of chlorophyll, was assayed in the replication of poliovirus (PV-1) and bovine herpesvirus (BoHV-1) in HEp-2 cell cultures.
Methods and Results:  Virucidal activity of CHLN was evaluated and the time-of-addition assay was performed as follows: before the infection (−1 and −2 h), at the time of the infection (0 h) and after the infection (1 and 2 h). Plaque reduction assay (PRA) showed that CHLN inhibited BoHV-1 and PV-1 infection and the 50% inhibitory concentrations (IC₅₀) against BoHV-1 and PV-1 infection were 8·6 and 19·8 μg ml⁻¹, respectively. The time-of-addition study demonstrated that the CHLN was effective inhibiting viral replication in 51% and 66·5% for PV-1 and BoHV-1, respectively, at the highest concentration of 20·0 μg ml⁻¹, when added during the infection. The directed effect of CHLN on viral strains demonstrated an inhibition of 62% and 66·4% for PV-1 and BoHV-1, respectively, by PRA.
Conclusions:  These results demonstrated that CHLN could be used as an antiviral suggesting directed activity on virus particles and on virus-receptor sites to BoHV. For poliovirus, CHLN also demonstrated virucide activity, moreover, showed to inhibit early steps of the replication cycle.
Significance and Impact of the Study:  CHLN demonstrated promising selectivity index for both virus strains; therefore, it can be used for the development of an antiviral agent.     

Mutagenicity of streptozotocin and several other nitrosourea compounds in Salmonella typhimurium.

The following nitrosourea compounds were compared for their ability to induce mutation (to histidine independence) in the histidine-requiring auxotroph Salmonella typhimurium his G46: MNU, streptozotocin (SZ, streptozocin) and its analogs SZA1 and SZA2, and the antitumor drugs BCNU, CCNU and DCNU. At equitoxic doses SZ, SZA1, SZA2 and MNU were almost equally mutagenic causing 150, 42, 140 and 170 mutants/106 survivors at 20% lethal dose (ID20) ALTHOUGH, ON A WIEGHT BASIS, SZ was the most mutagenic of all the compounds tested. At ID20 BCNU, CCNU and DCNU gave about 0.5 mutants/106 survivors. Our results show that these nitrosoureas, in common with many other drugs (such as cyclophosphamide, daunomycin, etc.) used in cancer chemotherapy, are highly mutagenic. The implication of our results in the screening of drugs for their mutagenicity to man is discussed.     

Mutagenicity and cytotoxicity of congeners of two classes of nitroso compounds in Chinese hamster ovary cells

The induction of mutation by certain nitrosamidines and nitrosamides has been quantitated utilizing the hypoxanthine--guanine phosphoribosyl transferase (HGPRT) locus in Chinese hamster ovary cells. Dose--response relationships for cytotoxicity and mutagenicity are presented for N-methyl-N-nitrosourea (MNU), N-ethyl-N-nitrosourea (ENU), N-butyl-N-nitrosourea (BNU), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), and N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG). Based on the concentration of each agent required to kill 90% of the cells, the following order of cytotoxicity was observed: MNNG greater than ENNG greater than MNU greater than ENU greater than BNU. This is the same order of potency as observed for mutation induction per unit concentration of mutagen.     

Differences between survival, mutagenicity and DNA replication in MMS- and MNU-treated V79 hamster cells

After treatment with methyl methanesulfonate (MMS) or N-methyl-N-nitrosourea (MNU), the mutagenicity and survival of Chinese hamster V79 cells were investigated, as well as the inhibition of daughter DNA synthesis and, using the DNA unwinding technique and hydroxylapatite chromatography, the character of the newly synthesized DNA was studied. It was found that different cytotoxicity and mutagenicity of MMS and MNU was accompanied by different types of DNA synthesis inhibition. The treatment with the former compound resulted in a longer inhibition of DNA synthesis, while the treatment with the latter showed that as early as 2 h after exposure the percentage of nascent DNA increased. Shortly after the exposure to both alkylating agents, the newly synthesized DNA contained a higher number of gaps than control DNA, in dependence on the concentration used. During culturing after treatment, the character of nascent DNA in MMS-treated cells gradually returned to that of control DNA, while MNU-treated cells, for the whole time of our study, synthesized DNA with a larger number of gaps than control DNA. We suggest that the character of nascent daughter DNA reflects the occurrence of lesions in parental DNA. These are repaired within a shorter time in MMS- than in MNU-treated cells. The long-term persistence of lesions in the DNA of MNU-treated cells might be one of the factors responsible not only for the higher cytotoxic but also for the many times higher mutagenic effect of this alkylating agent.     

Evaluation of the mutagenicity of sesquiterpenic compounds and their influence on the susceptibility towards antibiotics of two clinically relevant bacterial strains

Sesquiterpenic compounds are natural chemicals present in organisms from different Phylae or Divisions, which have proved to be important bioactive products, namely in potentiating the action of antibiotics. In the first step, the mutagenicity of nine sesquiterpenic compounds (hydrocarbons and alcohols) was screened in a Salmonella typhimurium his(-)-reversion test with strains TA98 and TA100, in the presence or absence of in vitro metabolic activation. Under the test conditions, none of the compounds showed mutagenicity up to a concentration of 222μg/plate. trans-Farnesol, nerolidol, and α-bisabolol displayed cytotoxicity when tested at concentrations ranging from 14 to 222μg/plate. Then, the combined effect of antibiotic-sesquiterpenic compounds was evaluated on two clinically relevant pathogens, Escherichia coli and Staphylococcus aureus, with well-defined resistance-sensitive profiles. The agar-disc diffusion assay revealed that all the combinations of antibiotic-sesquiterpenic compounds increased the antibacterial activity of the antibiotics tested against S. aureus. For E. coli, an antagonistic effect was observed for various combinations on the growth of this bacterium.