Modulation of mustard toxicity by tacrine.

Compounds containing the chloroethyl group are potent inhibitors of DNA synthesis and cell growth. Tacrine, a choline carrier inhibitor, was found to protect both HeLa cells and rat thymocytes against the effects of nitrogen mustard. DNA synthesis was restored from 13 to 71% of the control value and cell viability restored from 27 to 57% of the control value by exposure of the cells to an equimolar concentration of tacrine immediately prior to nitrogen mustard. In contrast, tacrine was unable to significantly protect rat thymocytes against the toxic effects of sulphur mustard. These results have implications for the clinical use of nitrogen mustard.

Allelic frequencies of p53 codon 72 polymorphism and human papillomavirus-mediated cervical cancer In Papua New Guinean women

Cervical cancer is regarded as a sexually transmitted disease caused by the human papilloma virus (HPV) detected in up to 80 per cent of the cancer biopsies. Genetic susceptibility of a p53 allelic variant has been postulated to play a vital role in carcinogenesis. This study was aimed at determining the allelic frequencies of p53 codon 72 polymorphism in Papua New Guinean women and also assessing the presence of HPV in cervical cancer biopsies. Peripheral blood (3-5 mL) was collected from 53 healthy females of reproductive age (19-37 years) with no known past and current history of HPV infections. Sixty-two cervical biopsies along with cervical swaps were obtained from patients (19-54 years) with clinical symptoms and histopathological confirmation of cervical cancer. DNA was extracted from the peripheral blood samples and cervical samples. Exon 4 was amplified with PCR and further genotypic analyses performed by Restriction fragment length polymorphism (RFLP) and single-stranded conformational polymorphism (SSCP). Of the 53 normal samples analyzed, 3.8 % (2/53) were Arginine homozygous, 58.5 % were Proline homozygous and 37.7 % were heterozygous. For the cancer samples, 14.5 % (9/62) were Arginine homozygous, 54.8 % were Proline homozygous and 30.7% were heterozygous. HPV genome was detected in 83.9 % (52/62) of the cervical cancer samples. The genotypic trend and allelic frequencies were consistent with literature.

The disposition of oral amodiaquine in Papua New Guinean children with falciparum malaria.

AIMS: We assessed the disposition of oral amodiaquine (AQ) and CYP2C8 polymorphism in 20 children with falciparum malaria. METHODS: AQ and DEAQ concentrations were determined with SPE-HPLC method. CYP2C8 genotypes were assessed by PCR-RFLP method. RESULTS: AQ was not detectable beyond day 3 postdose. Cmax for DEAQ was reached in 3.0 days. The mean values for t1/2, MRT, and AUCtotal were 10.1 days, 15.5 days and 4512.6 microg l(-1) day, respectively. All the children were CYP2C8* homozygous. CONCLUSION: Our data are consistent with those previously reported, and the AQ regimen seems pharmacokinetically adequate in the absence of CYP2C8 polymorphism.

Nitrogen mustard inhibits transcription and translation in a cell free system.

Nitrogen mustard and its derivatives such as cyclophosphamide, chlorambucil and melphalan are widely used anti-cancer agents, despite their non-specific reaction mechanism. In this study, the effect of alkylation by nitrogen mustard of DNA and RNA (coding for a single protein) was investigated using both a translation system and a coupled transcription/translation system. When alkylated DNA was used as the template for coupled transcription and translation, a single translation product corresponding to the 62 kDa luciferase protein was synthesised. Production of the translated product encoded by this template was inhibited by mustard concentrations as low as 10 nM, and 50% inhibition occurred with 30 nM mustard. A primer extension assay employed to verify alkylation sites on the DNA revealed that all guanine residues on the DNA template are susceptible to alkylation by nitrogen mustard. Similarly, when alkylated RNA was used as the template for protein synthesis, the amount of the 62 kDa luciferase protein decreased with increasing mustard concentration and a range of truncated polypeptides was synthesised. Under these conditions 50% inhibition of translation occurred with approximately 300 nM mustard (i.e. approximately 10 times that required for similar inhibition using an alkylated DNA template). Furthermore, a gel mobility shift assay revealed that mustard alkylation of the RNA template results in the formation of a more stable retarded RNA complex. The functional activity of the luciferase protein decreased with alkylation of both the DNA and RNA templates, with a half-life of loss of activity of 1.1 h for DNA exposed to 50 nM mustard, and 0.5 h for RNA exposed to 50 microM mustard. The data presented support the notion that DNA is a critical molecule in the mode of action of mustards.

Molecular basis of nitrogen mustard effects on transcription processes: role of depurination.

DNA was alkylated with nitrogen mustard (HN2) and the rate of release of the alkylpurines was quantitated by HPLC. The half life of depurination of the major product (7-alkylguanine) was 9.1 h at 37 degrees C. End-labelled DNA was used to show that depurination occurred dominantly at 5'-GA, 5'-GG and 5'-GT sequences. Although extensive alkylation was observed at all 5'-GNC and 5'GNT sequences, no depurination was observed at these sites during a depurination time of 20 h at 37 degrees C. Since these sites are potential interstrand crosslinking sequences (G-adduct-G and G-adduct-A, both spanning an intervening base pair), this suggests that these regions have a greatly enhanced stability or that simultaneous depurination of both ends of the crosslink is necessary before these lesions are removed (with a predicted half-life of approximately 80 h at 37 degrees C). Depurination at the lac UV5 promoter impaired the association of Escherichia coli RNA polymerase with that promoter, while in the elongation phase two distinctly different sequence-specific processes were apparent. At 5'-GNC and 5'-GNT sequences transcriptional blockages were maintained with increasing elongation time, whereas at monoadduct sites, the blockage decreased with elongation time (predominantly at 5'-GG and 5'-GC sequences), with an average half-life of approximately 10.7 h. Collectively, these results suggest that the observed read-through past monoadduct sites is due to depurination of the DNA at those sites. E. coli RNA polymerase is therefore able to transcribe efficiently past apurinic sites and presumably does so by incorporating an incorrect base into the nascent RNA.

Effect of sulphur mustard on the initiation and elongation of transcription.

Sulphur mustard is a potent alkylating agent that causes severe vesication as well as systemic and genotoxic effects. Despite its long history as a chemical warfare agent, the mechanism of its toxicity remains unknown and no successful pharmacological intervention has yet been found. In this study we have examined the effects of mustard alkylation of DNA on transcriptional processes. Gel mobility shift analysis shows that mustard alkylation of the lac UV5 promoter increases the stability of the promoter-RNA polymerase binary complex. Following formation of the initiation complex and addition of elongation nucleotides, approximately 45% of the RNA polymerase in the initiated complex remained associated with the alkylated promoter, compared to only 7% remaining associated with the unalkylated promoter. For the RNA polymerase able to escape the initiation complex, mustard alkylation of the DNA template resulted in the production of truncated transcripts. Analysis of these truncated transcripts revealed that sulphur mustard alkylates DNA preferentially at 5'-AA, 5'-GG and 5'-GNC sequences on the DNA template strand and this is significantly different from the alkylation sites observed with nitrogen mustard. This study represents the first report at the molecular level of sulphur mustard-induced effects on transcriptional processes.