CHLOROPHYLLIN IS BOTH A POSITIVE AND NEGATIVE MODIFIER OF MUTAGENICITY

The mechanism responsible for the modification of mutagenicity by chlorophyllin has been investigated using mutagenic compounds with different mechanisms of action, including the monofunctional alkylating agents, N-methyl-N'-nitrosourea (MNU) and ethylmethanesulphonate (EMS); nitrosamines related to tobacco products, i.e. dimethyl-nitrosamine (DMN), N-nitrosonornicotine (NNN) and 4-(N-methyl-N-nitrosoamino)-1-(3-pyridinyl)-2-butanone (NNK); the polycyclic aromatic hydrocarbon (PAH) benzo[a]pyrene (B[a]P) and two of its metabolites, i.e. (-)-7beta,8alpha-dihydroxy-7, 8-dihydrobenzo[a]pyrene (7,8-diol) and (+)-7beta,8alpha-dihydroxy-9alpha, 10alpha-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE); and a complex mutagenic mixture, an extract and subfractions of Swedish moist oral snuff (SMOS). Mutagenicity was monitored with the Ames Salmonella/microsome assays (STY) and hprt V79 point mutation assay (V79). The effects of chlorophyllin on the mutagenicity of the nitrosamines in the STY assays were found to be complex. In the presence of either NNN or NNK, low concentrations of chlorophyllin actually potentiated the mutagenicity >2-fold. However, at higher, but still non-toxic concentrations, chlorophyllin decreased the mutagenicity of both compounds. The same type of dose - response relationship for chlorophyllin was indicated in the V79 assay system with DMN, although the effect was much weaker. The results with STY were further confirmed by replacing chlorohylin with another porphyrin compound, hemin. In contrast, biliverdin, a porphyrin structure without the central metal ion, was unable to potentiate the mutagenicity of NNK in STY. These findings suggest that chlorophyllin interferes with electron transport, which was further supported by the finding that glucose-6-phosphate can be excluded from the bioactivating NADPH-generating S9 mixture in the presence of chlorophyllin without altering the mutagenicity. The mutagenicity of MNU or EMS was not influenced in either of the assays, which is consistent with the hypothesis that the modification of nitrosamine mutagenicity may involve changes in metabolic activation. The potentiating effect of chlorophyllin was further investigated using both B[a]P and BPDE in the STY assay and 7,8-diol and BPDE in the V79 assay. The results show a complete supression of the mutagenicity of all three compounds in both systems by non-toxic levels of chlorophyllin. There was no enhancement of PAH-induced mutations with low concentrations of chlorophyllin. Since enhancement only occurs with nitrosamines we suggest that chlorophyllin specifically interferes with the form of cytochrome P450 catalyzing DMN-demethylase activity. Findings with the complex mixtures SMOS were similar to these with PAH, indicating that the major mutagenic activity in these extracts is not caused by nitrosamines. To date, only speculations, about the mechanism by which chlorophyllin exerts its antimutagenic activity have been presented. Scavenging of radicals or supression of metabolic activation, as well as inactivation due to complex formation with either the metabolically activated mutagen or a non-activated precursor, have all been suggested. We conclude that chlorophyllin has several mechanisms of action, including anti-mutagenic activity arising from complex formation with PAH; no effect on monofunctional alkylating agents; and, finally, interference with electron transport to a specific form of cytochrome P450.