Stability of benzo[a]pyrene DNA adducts in rat tissues during their long-term storage at − 20°C or − 80°C

Abstract

The aim of this study was to examine whether the storage of tissues at − 80°C or − 20°C affects the benzo[a]pyrene(B[a]P)-derived DNA adduct pattern and levels in rat tissues. Three rats were treated orally with a single dose of 100 mg B[a]P/kg b.w. and killed 24 h later. White blood cells (WBC) were isolated from the fresh blood. Livers, lungs and hearts were immediately removed, dissected into small fragments and were pooled for each organ. Pooled samples were proportionally divided into 7 aliquots. DNA from the first aliquot was immediately isolated (time 0). The other aliquots were frozen and stored at − 20°C or at − 80°C. DNA was isolated from the frozen samples at 1, 5 and 10 months later. ³²P-postlabeling analysis was performed at the beginning and at the end of study with the whole set of samples. Two B[a]P-derived adducts were detected in all tissues but with different intensities types. One of the adducts was found predominantly in WBC (∼ 85%) and liver (∼ 68%), while heart and lung accounted only for ∼ 43% and ∼ 39%, respectively. This adduct was tentatively identified as benzo[a]pyrene diol-epoxide-N² adduct (BPDE-N²-dG) based on TLC and HPLC analyses of ³²P-postlabeled adducts. The highest total DNA adduct level (sum of 2 spots) was found in lung (4.90 adducts/10⁸ nucleotides) compared with heart, liver and WBC (3.55, 2.37 and 2.32 adducts/10⁸ nucleotides, respectively). The analysis of variance provided evidence that storage of tissues at − 20°C or at −80°C up to 10 months did not significantly affect B[a]P DNA adduct levels and patterns in the rat lung, heart and liver. Our study indicates that properly stored tissues can be used for DNA adduct analysis with confidence.