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Alkylating agents are present in food and tobacco smoke, but are also used in cancer chemotherapy, inducing the DNA lesion O6-methylguanine.This critical adduct is repaired by O6-methylguanine-DNA methyltransferase (MGMT), resulting in MGMT inactivation and degradation. In the present study, we analyzed the effects of the natural disulfide compound lipoic acid (LA) on MGMT in vitro and in colorectal cancer cells. We show that LA, but not its reduced form dihydrolipoic acid, potently inhibits the activity of recombinant MGMT by interfering with its catalytic Cys-145 residue, which was partially reversible by N-acetyl cysteine. Incubation of HCT116 colorectal cancer cells with LA altered their glutathione pool and caused a decline in MGMT activity. This was mirrored by LA-induced depletion of MGMT protein, which was not attributable to changes in MGMT messenger RNA levels. Loss of MGMT protein coincided with LA-induced autophagy, a process resulting in lysosomal degradation of proteins, including presumably MGMT. LA-stimulated autophagy in a p53-independent manner as revealed by the response of isogenic HCT116 cell lines. Knockdown of the crucial autophagy component beclin-1 and chemical inhibitors blocked LA-induced autophagy, but did not abrogate LA-triggered MGMT degradation. Concomitant with MGMT depletion, LA pretreatment resulted in enhanced O6-methylguanine levels in DNA. It also increased the cytotoxicity of the alkylating anticancer drug temozolomide in temozolomide-resistant colorectal cancer cells. Taken together, our study showed that the natural compound LA inhibits MGMT and induces autophagy. Furthermore, LA enhanced the cytotoxic effects of temozolomide, which makes it a candidate for a supplement in cancer therapy. [ABSTRACT FROM AUTHOR]

Hyperbaric oxygen (HBO) treatment of human subjects (i.e. exposure to 100% oxygen at a pressure of 2.5 ATA for a total period of 3 × 20 min) caused clear and reproducible DNA damage in lymphocytes, as detected with the comet assay (single cell gel electrophoresis). Induction of DNA damage was found only after the first HBO exposure and not after further treatments of the same individuals. Furthermore, blood taken 24 h after HBO treatment was significantly protected against the induction of DNA damage by hydrogen peroxide (H2O2) in vitro, indicating that adaptation occurred due to induction of antioxidant defenses. The cells were not significantly protected against the genotoxic effects of γ-irradiation, suggesting increased scavenging of reactive oxygen species distant from nuclear DNA or an inducible change in the levels of free transition metals. We now demonstrate increased levels of heme oxygenase-1 (HO-1) in lymphocytes 24 h after HBO treatment of volunteers. Under the same conditions, superoxide dismutase, catalase and the DNA repair enzymes apurinic endonuclease and DNA polymerase β were not enhanced in expression. We also show that protection against the induction of DNA damage by H2O2 in lymphocytes even occurs with a shortened HBO treatment which did not induce significant DNA damage by itself. Our results suggest that increased sequestration of iron as a consequence of induced HO-1 might be involved in the adaptive protection after HBO treatment and that the induction of DNA damage is not the trigger for adaptive protection. [ABSTRACT FROM PUBLISHER]

Using the multistage (initiation-conversion-promotion) protocol we have studied the effects of methyl methane-sulfonate (MMS), a well-known alkylating and clastogenic agent, on tumor development in NMRI mouse skin . When topically applied in a dose up to 400 μmol MMS did not exhibit any initiating efficacy while under identical conditions chromosomal damage (mainly breaks and gaps) was induced in epidermis. A dose of 100 μmol MMS was found to be almost as clastogenic as 10 μmol of the convertogenic tumor promoter 12--tetradecanoylphorbol-13-acetate (TPA), whereas the non-convertogenic promoter 12--retinoylphorbol-13-acetate (TPA, 10 nmol) did not induce chromosomal aberrations . In combination with initiation by 7,12-dimethylbenz[a]anthracene (DMBA) and promotion by RPA, MMS (2 × 100 μmol) turned out to be a rather powerful convertogenic agent (‘stage I tumor promoter’). This tumor-inducing efficacy of MMS was synergistically increased by simultaneous application of RPA. These results support the concept that the induction of chromosomal aberrations plays an important role in skin tumor development, i.e. in the conversion stage. [ABSTRACT FROM PUBLISHER]

Chinese hamster cells of the mutant strain W27-1 which is hypersensitive to UV and monofunctional alkylating agents were transfected with human DNA ligated to the bacterial xanthine-guanine phosphoribosyltransferase (gpt) gene. Selection was performed for resistance to mycophenolic acid and finally for survival after treatment with high doses of methyl methanesulfonate. A gpt transfectant was generated (T38-2-7) which acquired resistance to methyl methanesulfonate and cross-resistance to -methyl-′ -nitro--nitrosoguanidine at levels comparable with the parental (wild-type) strain CHO-9. T38-2-7 cells were not more resistant, however, to UV, mitomycin C and -hydroxyethyl--chloroethylnitrosourea than the mutant W27-1. The transfectant contains integrated human DNA and was shown to be deficient for the O methyltransferase. The results indicate that the transfected DNA specifically complemented the defect underlying alkylation hypersensitivity of W27-1 cells or that a gene was transfected which is generally inactive in CHO cells and which causes alkylation resistance. [ABSTRACT FROM PUBLISHER]

1,3-Dimethyl-3-phenyl-1-nitrosourea (DMPNU) shows no spontaneous decomposition in aqueous buffered solution (pH 7.0) at 37°C over a measuring period of 10 days, but it is directly genotoxic in all assays applied. This compound induces base substitutions in Salmonella typhimurium (especially in the plasmid harbouring strain TA 100), gene mutations at the HGPRT locus of Chinese hamster V79 cells, and is a very potent inducer of clastogenic damage and sister chromatid exchanges in V79 cells. Data on gene mutation induction by 1-methyl-1-nitrosourea are included for comparison. The findings on DMPNU are discussed in comparison with trialkylnitrosoureas. In contrast to these latter compounds, DMPNU is active without addition of S9 mix . It is asswned that its genotoxc activity is due to an intracellular catalytic degradation favoured by the electron- withdrawing effect of the phenyl group. [ABSTRACT FROM PUBLISHER]