Chloroethylnitrosoureas (CENUs) are an important type of alkylating agent employed in the clinical treatment of cancer. However, the anticancer efficacy of CENUs is greatly decreased by a DNA repairing enzyme, O6-alkylguanine-DNA alkyltransferase (AGT), by preventing the formation of interstrand cross-links (ICLs). In this study, a combi-nitrosourea prodrug, namely, N-(2-chloroethyl)-N′-2-(O6-benzyl-9-guanine)ethyl-N-nitrosourea (BGCNU), which possesses an O6-benzylguanine (O6-BG) derivative and CENU pharmacophores simultaneously, was synthesized and evaluated for its ability to induce ICLs. The target compound is markedly more cytotoxic in human glioma cells than the clinically used CENU chemotherapies ACNU, BCNU, and their respective combinations with O6-BG. In the AGT-proficient cells, significantly higher levels of DNA ICLs were observed in the groups treated by BGCNU than those by ACNU and BCNU, which indicated that the activity of AGT was effectively inhibited by the O6-BG derivatives released from BGCNU.Keywords: AGT inhibition; anticancer activity; chloroethylnitrosourea; Combi-nitrosourea; DNA interstrand cross-links;

Heavy metal contamination induced by cigarette smoking is a critical issue in environmental pollution and human health risk. Although a number of investigations have been performed on the detection of heavy metals in cigarette tobacco, only very limited data have been reported for the determination of these metals in other parts of cigarettes. In this study, the contents of copper, cadmium, chromium, nickel and lead in cigarette tobacco, smoke, ash and butts were quantified using high-resolution continuum source graphite furnace atomic absorption spectrometry. The LODs for Cu, Cd, Cr, Ni and Pb were 0.010 μg L−1, 0.020 μg L−1, 0.025 μg L−1, 0.030 μg L−1 and 0.012 μg L−1, respectively. Twelve cigarette samples purchased from eleven popular brands in China were used for the heavy metal measurements. Microwave digestion was employed for the preparation of the cigarette tobacco, ash and butt samples. A smoke collection apparatus, which was designed to simulate human smoking behavior by alternating suction and pause intervals, was used to capture the heavy metals in smoke. The total amounts of the determined metals were observed to be the highest in ash (55.60 to 125.99 μg g−1), followed by tobacco (11.70 to 22.34 μg g−1) and butts (4.29 to 15.22 μg g−1), and were 0.67, 0.10, 0.14, 0.16 and 0.24 μg per cigarette for Cu, Cd, Cr, Ni and Pb, respectively, in smoke. The residual rates of the heavy metals in ash, smoke and butts were measured to estimate the distribution of each element after the cigarette tobacco was fully burnt. The results indicated that the majority of the heavy metals (41.1% to 68.7%) remained in ash, with 10.0% to 28.0% condensed in butts and 6.6% to 22.9% released into the mainstream smoke. Moreover, a high variance of the five metal contents was observed in different brands of cigarettes, which might be associated with the geographical origin of the tobacco plant, cigarette production procedure, and transportation and storage of the materials and products. This study provides a reliable method for the quantitation of heavy metals in different components of cigarettes, which will be beneficial to the prevention of heavy metal hazards and the reduction of human health risks through tobacco consumption.

In the presence of sodium carbonate, the [4 + 2] cycloaddition of α-halogeno hydrazones to imines proceeded readily, and furnished 2,3,4,5-tetrahydro-1,2,4-triazines in moderate to high chemical yields.

•A combi-nitrosourea prodrug releasing DNA cross-linking agent and AGT inhibitor was synthesized.•The combi-nitrosourea exhibits higher cytotoxicity against mer+ glioma cells than ACNU.•The prodrug induces higher levels of dG–dC crosslink than traditional CENU chemotherapies.•AGT activity is efficiently inhibited by the O6-benzylguanine analog released from the prodrug.The drug resistance of CENUs induced by O6-alkylguanine-DNA alkyltransferase (AGT), which repairs the O6-alkylated guanine and subsequently inhibits the formation of dG–dC cross-links, hinders the application of CENU chemotherapies. Therefore, the discovery of CENU analogs with AGT inhibiting activity is a promising approach leading to novel CENU chemotherapies with high therapeutic index. In this study, a new combi-nitrosourea prodrug 3-(3-(((2-amino-9H-purin-6-yl)oxy)methyl)benzyl)-1-(2-chloroethyl)-1-nitrosourea (6), designed to release a DNA cross-linking agent and an inhibitor of AGT, was synthesized and evaluated for its antitumor activity and ability to induce DNA interstrand cross-links (ICLs). The results indicated that 6 exhibited higher cytotoxicity against mer+ glioma cells compared with ACNU, BCNU, and their respective combinations with O6-benzylguanine (O6-BG). Quantifications of dG–dC cross-links induced by 6 were performed using HPLC–ESI-MS/MS. Higher levels of dG–dC cross-link were observed in 6-treated human glioma SF763 cells (mer+), whereas lower levels of dG–dC cross-link were observed in 6-treated calf thymus DNA, when compared with the groups treated with BCNU and ACNU. The results suggested that the superiority of 6 might result from the AGT inhibitory moiety, which specifically functions in cells with AGT activity. Molecular docking studies indicated that five hydrogen bonds were formed between the O6-BG analogs released from 6 and the five residues in the active pocket of AGT, which provided a reasonable explanation for the higher AGT-inhibitory activity of 6 than O6-BG.

⿢An LC⿿MS/MS-based method for measuring AGT activity was established.⿢AGT activity can be accurately quantified by the nonradioactive method.⿢The novel method was applied for determining AGT activity in tumor cells.⿢The method is promising in evaluating drug resistance to chemotherapies.The repair of DNA mediated by O6-alkylguanine-DNA alkyltransferase (AGT) provides protection against DNA damage from endogenous or exogenous alkylation of the O6 position of guanine. However, this repair acts as a double-edged sword in cancer treatment, as it not only protects normal cells from chemotherapy-associated toxicities, but also results in cancer cell resistance to guanine O6-alkylating antitumour agents. Thus, AGT plays an important role in predicting the individual susceptibility to guanine O6-alkylating carcinogens and chemotherapies. Accordingly, it is necessary to establish a quantitative method for determining AGT activity with high accuracy, sensitivity and practicality. Here, we describe a novel nonradioactive method for measuring AGT activity using stable isotope dilution high-performance liquid chromatography electrospray ionization tandem mass spectrometry (HPLC-ESI⿿MS/MS). This method is based on the irreversibility of the removal of the O6-alkyl group from guanine by AGT and on the high affinity of O6-benzylguanine (O6-BG) as an AGT substrate. HPLC-ESI⿿MS/MS was used to measure the AGT activities in cell protein extracts from eight tumour lines, demonstrating that AGT activity was quite variable among different cell lines, ranging from nondetectable to 1021 fmol/mg protein. The experiments performed in intact tumour cells yielded similar results but exhibited slightly higher activities than those observed in cell protein extracts. The accuracy of this method was confirmed by an examination of AGT expression levels using western blotting analysis. To our knowledge, this method is the first mass spectrometry-based AGT activity assay, and will likely provide assistance in the screening of cancer risk or the application of chemotherapies.

Chloroethylnitrosoureas (CENUs) are bifunctional alkylating agents widely used for the clinical treatment of cancer. They exert anticancer activity by inducing DNA interstrand cross-links (ICLs) within GC base pairs to form dG-dC cross-links. This lesion inhibits DNA double strand separation during replication and transcription and results in the apoptosis of cancer cells. However, O6-alkylguanine DNA alkyltransferase (AGT) repairs the DNA ICLs by removing the alkyl group at the O6 position of either O6-(2-chloroethyl)deoxyguanosine (O6-ClEtdGuo) or N1,O6-ethanodeoxyguanosine (N1,O6-EtdGuo), which are intermediates in the formation of dG-dC cross-links. The action of AGT leads to drug resistance against CENUs. O6-Benzylguanine (O6-BG) was identified as an effective AGT inhibitor that enhances the antitumor effects of CENUs. In this study, the effect of O6-BG on the formation of dG-dC cross-links was investigated by treating human brain glioma SF767 cells with 1-[(4-amino-2-methyl-5-pyrimidinyl)methyl]-3-(2-chloroethyl)-3-nitrosourea (ACNU). The levels of dG-dC cross-link were determined using stable isotope dilution high-performance liquid chromatography electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS). The results indicated that ACNU induced higher levels of dG-dC cross-link in SF767 cells pretreated with O6-BG compared to cells without O6-BG pretreatment. The highest dG-dC cross-linking levels were generally observed at 12 h for all drug concentration groups, a result which was consistent with cytotoxicity assay. These results provided direct evidence for the enhancement of dG-dC cross-linking levels caused by the inhibition of AGT by O6-BG. These data indicate that dG-dC cross-links may be developed as a biomarker for evaluating the activity of novel O6-BG analogues as AGT inhibitors for combination therapy with CENUs.

•DNA interstrand crosslinks induced by CENUs were determined using HPLC–MS/MS.•The crosslinking activities of 4 types of CENU were compared.•The crosslinking level induced by ACNU is much higher than the other 3 CENUs.•A correlation was found between crosslinking level and anticancer efficiency.•G–C crosslinking level is a potential biomarker for anticancer activity of CENUs.Chloroethylnitrosoureas (CENUs) are an important family of alkylating agents employed in the clinical treatments of cancer. They exert cytotoxicity by inducing DNA interstrand crosslinks (ICLs) between guanine and the complimentary cytosine, namely dG–dC crosslink. Many investigations have been performed on the DNA ICLs involved in the anticancer efficacy of CENUs, but no conclusive comparisons between these agents have been published. In this work, the levels of dG–dC crosslink in calf thymus DNA induced by four CENUs, including nimustine (ACNU), carmustine (BCNU), lomustine (CCNU) and fotemustine (FTMS), were quantitatively determined using HPLC–ESI-MS/MS. The obtained time-courses for the dG–dC crosslinking levels indicated that there is an induction period with very low crosslinking activity at the initial stage of the treatment by BCNU and CCNU. The induction period provides a convincing evidence for the presumed mechanism that the formation of dG–dC crosslinks was initiated by the monoalkylation of guanine followed by the second alkylation of the complimentary cytosine. The crosslinking activity of ACNU is remarkably higher than those of BCNU, CCNU and FTMS at all time points. The crosslinking activities of CENUs were found to be related to their stability in aqueous solution. ACNU has the shortest half-life among the four CENUs, but has highest crosslinking levels; on the contrary, CCNU has the lowest crosslinking activity with the longest half-life. Moreover, a correlation was found between the crosslinking activity and the anticancer efficiency. ACNU with the highest crosslinking activity showed the better survival gain for high-grade glioma than BCNU, CCNU and FTMS as reported in an epidemiological study. This suggests that dG–dC crosslink can possibly be employed as a potential biomarker for evaluating the anticancer efficiency of novel CENU drugs.

DNA phosphotriester adducts are common alkylation products of DNA phosphodiester moiety induced by N-nitrosoureas. The 2-hydroxyethyl phosphotriester was reported to hydrolyze more rapidly than other alkyl phosphotriesters both in neutral and in alkaline conditions, which can cause DNA single strand scission. In this work, DFT calculations have been employed to map out the four lowest activation free-energy profiles for neutral and alkaline hydrolysis of triethyl phosphate (TEP) and diethyl 2-hydroxyethyl phosphate (DEHEP). All the hydrolysis pathways were illuminated to be stepwise involving an acyclic or cyclic phosphorane intermediate for TEP or DEHEP, respectively. The rate-limiting step for all the hydrolysis reactions was found to be the formation of phosphorane intermediate, with the exception of DEHEP hydrolysis in alkaline conditions that the decomposition process turned out to be the rate-limiting step, owing to the extraordinary low formation barrier of cyclic phosphorane intermediate catalyzed by hydroxide. The rate-limiting barriers obtained for the four reactions are all consistent with the available experimental information concerning the corresponding hydrolysis reactions of phosphotriesters. Our calculations performed on the phosphate triesters hydrolysis predict that the lower formation barriers of cyclic phosphorane intermediates compared to its acyclic counter-part should be the dominant factor governing the hydrolysis rate enhancement of DEHEP relative to TEP both in neutral and in alkaline conditions.

The contents of lead, cadmium, chromium, copper, and nickel were determined in 25 tea samples from China, including green, yellow, white, oolong, black, Pu'er, and jasmine tea products, using high-resolution continuum source graphite furnace atomic absorption spectrometry. The methods used for sample preparation, digestion, and quantificational analysis were established, generating satisfactory analytical precisions (represented by relative standard deviations ranging from 0.6% to 2.5%) and recoveries (98.91–101.32%). The lead contents in tea leaves were 0.48–10.57 mg/kg, and 80% of these values were below the maximum values stated by the guidelines in China. The contents of cadmium and chromium ranged from 0.01 mg/kg to 0.39 mg/kg and from 0.27 mg/kg to 2.45 mg/kg, respectively, remaining in compliance with the limits stipulated by China's Ministry of Agriculture. The copper contents were 7.73–63.71 mg/kg; only 64% of these values complied with the standards stipulated by the Ministry of Agriculture. The nickel contents ranged from 2.70 mg/kg to 13.41 mg/kg. Consequently, more attention must be paid to the risks of heavy metal contamination in tea. The quantitative method established in this work lays a foundation for preventing heavy metal toxicity in human from drinking tea and will help establish regulations to control the contents of heavy metals in tea.