Temozolomide is a DNA-methylating agent used in cancer chemotherapy, notably for glioblastoma multiforme (GBM) where it is applied as a front-line drug. One of the DNA alkylation products is the minor lesion O6-methylguanine (O6MeG), which is responsible for nearly all genotoxic, cytotoxic and cytostatic effects of TMZ in the low-dose range relevant for cancer therapy.Here, we addressed the question of how many O6MeG adducts are required to elicit cytotoxic responses. Adduct quantification revealed that O6MeG increases linearly with dose. The same was observed for DNA double-strand breaks (DSBs) and p53ser15. Regarding apoptosis, hockeystick modeling indicated a possible threshold for A172 cells at 2.5 µM TMZ, while for LN229 cells no threshold was detected. Cellular senescence, which is the main cellular response, also increased linearly, without a threshold. Using a dose of 20 µM, which is achievable in a therapeutic setting, we determined that 14,000 adducts give rise to 32 DSBs (γH2AX foci) in A172 cells. This leads to 12% cell death and 35% of cells entering senescence. In LN229 cells, 20 µM TMZ induced 20,600 O6MeG adducts, 66 DSBs (γH2AX foci), 24% apoptosis and 52% senescence. The linear dose-response and the genotoxic and cytotoxic effects observed at therapeutically relevant dose levels makes it very likely that the TMZ target concentration triggers a significant cytotoxic and cytostatic effect in vivo. Despite a linear increase in the O6MeG adduct level, DSBs and p53 activation, the low curative effect of TMZ results presumably from the low rate of apoptosis compared to senescence.

N-nitroso compounds are alkylating agents, which occur widespread in diet and environment. They induce DNA alkylation adducts such as O 6-methylguanine (O 6-MeG), which is repaired by O 6-methylguanine-DNA methyltransferase (MGMT). Persistent O 6-MeG lesions have detrimental biological consequences like mutagenicity and cytotoxicity. Due to its pivotal role in the etiology of cancer and in cytotoxic cancer therapy, it is important to detect and quantify O 6-MeG in biological specimens in a sensitive and accurate manner. Here, we used immunological approaches and established an ultra-performance liquid chromatographytandem mass spectrometry (UPLC-MS/MS) to monitor O 6-MeG adducts. First, colorectal cancer (CRC) cells were treated with the methylating anticancer drug temozolomide (TMZ). Immunofluorescence microscopy and an immuno-slot blot assay, both based on an adductspecific antibody, allowed for the semi-quantitative, dose-dependent assessment of O 6-MeG in CRC cells. Using the highly sensitive and specific UPLC-MS/MS, TMZ-induced O 6-MeG adducts were quantified in CRC cells and even in peripheral blood mononuclear cells exposed to clinically relevant TMZ doses. Furthermore, all methodologies were used to detect O 6-MeG in wildtype (WT) and MGMT-deficient mice challenged with the carcinogen azoxymethane. UPLC-MS/MS measurements and dose-response modeling revealed a nonlinear formation of hepatic and colonic O 6-MeG adducts in WT, whereas linear O 6-MeG formation without a threshold was observed in MGMT-deficient mice. Collectively, the UPLC-MS/MS analysis is highly sensitive and specific for O 6-MeG, thereby allowing for the first time for the determination of thresholds upon exposure to O 6-methylating agents. We envision that this method will be instrumental to monitor the efficacy of methylating chemotherapy and to assess dietary exposures. MATERIAL AND METHODS Material All solutions were made with deionized water (18.2 MΩ resistivity). O 6-methyl-d3-guanine (O 6-Me-d3-G) was from Toronto Research Chemicals (Toronto, Canada). O 6-MeG, ammonium hydroxide (ACS Reagent 28-30 %), hydrochloric acid (ACS Reagent 37 %), and acetic acid (HPLC grade) were purchased from Sigma-Aldrich (Buchs, Switzerland). Guanine was obtained from Acros Organics (New Jersey, USA) and Strata-X polymeric columns (30 μm) were from Phenomenex (Torrance, USA). Methanol (HPLC Gradient Grade) was bought at VWR (Dietikon, Switzerland).

We investigated how fibers in the anterior lateral line nerve of goldfish, Carassius auratus, respond to water motions generated by an object that was moved alongside the fish. Motion direction was from anterior to posterior or opposite, object diameter was between 0.1 and 4 cm and the distance between object and fish varied between 1 and 6 cm. Fibers exhibited monophasic responses characterized by a transient increase in discharge rate, biphasic responses consisting of an increase followed by a decrease in discharge rate or vice versa, or triphasic responses characterized by a rate increase followed by a decrease and again an increase or by the inverse pattern. In two-thirds of the fibers response patterns depended on object motion direction. Of these, about 60% responded to a reversal of motion direction with an inversion of the response pattern. Our results differ from previous data obtained from posterior lateral line nerve fibers in the relative proportions of the observed response patterns, and by a much smaller proportion of fibers that exhibited a direction-dependent response. These differences can be explained by the fact that the spatial orientations of the neuromasts on the head are more heterogenuous than on the trunk.

The O 6-alkylguanosine adduct O 6-carboxymethyldeoxyguanosine (O 6-CMdG) has been detected at elevated levels in blood and tissue samples from colorectal cancer patients and from healthy volunteers after consuming red meat. The diazo compound l-azaserine leads to the formation of O 6-CMdG as well as the corresponding methyl adduct O 6-methyldeoxyguanosine (O 6-MedG) in cells and is therefore in wide use as a chemical probe in cellular studies concerning DNA damage and mutation. However, there remain knowledge gaps concerning the chemical basis of DNA adduct formation by l-azaserine. To characterize O 6-CMdG formation by l-azaserine, we carried out a combination of chemical and enzymatic stability and reactivity studies supported by liquid chromatography tandem mass spectrometry for the simultaneous quantification of O 6-CMdG and O 6-MedG. We found that l-azaserine is stable under physiological and alkaline conditions as well as in active biological matrices but undergoes acid-catalyzed hydrolysis. We show, for the first time, that l-azaserine reacts directly with guanosine (dG) and oligonucleotides to form an O 6-serine-CMdG (O 6-Ser-CMdG) adduct. Moreover, by characterizing the reaction of dG with l-azaserine, we demonstrate that O 6-Ser-CMdG forms as an intermediate that spontaneously decomposes to form O 6-CMdG. Finally, we quantified levels of O 6-CMdG and O 6-MedG in a human cell line exposed to l-azaserine and found maximal adduct levels after 48 h. The findings of this work elucidate the chemical basis of how l-azaserine reacts with deoxyguanosine and support its use as a chemical probe for N-nitroso compound exposure in carcinogenesis research, particularly concerning the identification of pathways and factors that promote adduct formation.

A mong cancer patients the use of complementary and alternative medicine to treat disease-related depression, alleviate side effects, or even improve therapeutic efficacy varies between 29% in Europe to 87% in the US. 1,2 These medicines comprise supplements, vitamins, minerals and herbs, special foods, and diets as well as massage and spiritual therapy. While vitamins and minerals are the most common complementary and alternative medicines used by cancer patients, herbal supplements rank second. Half of the time, there may be a known risk for a drug interaction, but only around one-third of patients may inform their doctor about the use of these agents. 1 Some can contain pharmacologically active compounds with the potential to alter the bioavailability of cancer drugs by influencing, for example, drug-metabolizing enzymes or transporters.Interactions arising from concomitant use of bioactive alternatives and conventional chemotherapy could alter plasma levels of active drugs and influence therapeutic efficacy. 3,4 There are a large number of in vitro studies exploring the possible benefits of combining herbal components with anticancer agents, fewer in vivo studies, and very limited clinical data addressing efficacy and safety of combinations.Three illustrative examples include grapefruit juice, St. John's wort and chrysin. Highlighting these examples provides a viewpoint on the current understanding and underscores pharmacological concepts relevant to a range of combinations currently used by patients or that may arise with new therapeutics.