Preparation of a chemiluminescent imidoester for the non-radioactive labelling of proteins.

A chemiluminescent aryl acridinium ester was synthesized which possesses an imidate ester group capable of reacting with proteins under mild conditions. The compound can be detected at levels as low as 5.2 x 10(-19) mol using commercially available luminometers and can therefore be used to produce high specific activity labelled antibodies for use in immunochemiluminometric assays. The imidate ester compares favourably with a previously reported N-succinimidyl ester in terms of its labelling properties but is easier to synthesize, requiring one less step. The compound was used to label affinity purified to synthesize, requiring one less step. The compound was used to label affinity purified sheep antibodies to human parathyroid hormone to demonstrate its utility in a two-site immunochemiluminometric assay for the measurement of intact parathyroid hormone.

Toxicity and DNA damage induced by 1-nitropyrene and its derivatives in Chinese hamster lung fibroblasts.

1-Nitropyrene and its chemically synthesised derivatives were investigated for their cytotoxicity and ability to induce DNA-strand breaks in Chinese hamster lung fibroblasts. Both 1-nitrosopyrene (0.25-60 micrograms/ml) and 1-aminopyrene (0.25-25 micrograms/ml) were cytotoxic, and induced the formation of DNA lesions, which were measured as DNA single-strand breaks after sedimentation in alkaline sucrose-density gradients. Higher doses of 1-aminopyrene (25-60 micrograms/ml) inhibited the formation of DNA single-strand breaks. 1-Nitropyrene was not toxic (0.25-60 micrograms/ml) and induced low levels of detectable DNA strand breaks, whilst N-acetyl-1-aminopyrene was inactive. The post-mitochondrial supernatant fraction of Aroclor-induced rat-liver containing 4 mM NADPH (S9 mix) did not promote the activation of 1-nitropyrene. In fact DNA strand breaks induced by either 1-nitropyrene or 1-nitrosopyrene was abolished in the presence of S9 mix. The 1-nitropyrene reduced intermediate, N-hydroxy-1-aminopyrene was synthesised by the reduction of 1-nitrosopyrene with ascorbic acid. In the presence of ascorbic acid, 1-nitrosopyrene caused a 5-fold increase in the number of DNA single-strand breaks when compared to cells treated with 1-nitrosopyrene alone. The results are discussed in terms of the metabolic activation of 1-nitropyrene and 1-aminopyrene in Chinese hamster lung cells.

The induction of DNA adducts in mammalian cells exposed to 1-nitropyrene and its nitro-reduced derivatives.

1-Nitropyrene, 1-nitrosopyrene and 1-aminopyrene were investigated for their ability to induce covalently bound DNA adducts in calf thymus DNA and Chinese hamster lung fibroblasts. Xanthine oxidase catalysed the induction of one major and one minor DNA adduct in 1-nitropyrene- or 1-nitrosopyrene-treated calf thymus DNA, whilst 1-aminopyrene was inactive. These compounds did not form detectable DNA adducts in the absence of xanthine oxidase. The major DNA adduct produced by 1-nitropyrene and 1-nitrosopyrene in calf thymus DNA co-migrated on h.p.l.c., and the structure was consistent with that previously described by others as N-(deoxyguanosin-8-yl)-1-aminopyrene. The compounds were investigated for their ability to form DNA adducts in Chinese hamster lung fibroblasts. 1-Nitropyrene (5.2 pmol/mg DNA/h) and 1-nitrosopyrene (129 pmol/mg DNA/h) formed a single DNA adduct in Chinese hamster lung cells which co-eluted on h.p.l.c. with the C-8 deoxyguanosine adduct isolated from 1-nitropyrene-treated calf thymus DNA. 1-Nitrosopyrene was the most efficient compound investigated for the production of the C-8 guanine adducts. In contrast, 1-aminopyrene (14.7 pmol/mg DNA/h) induced the formation of a DNA adduct which did not co-elute with the C-8 guanine adduct. The data presented here suggest that 1-nitropyrene and 1-aminopyrene are metabolized to reactive intermediates which form different DNA adducts in Chinese hamster lung fibroblasts.