phosphorus-radioisotopes and hydroquinone

Our interest in benzene-DNA adduct formation and their consequence has led us to develop a number of sensitive methods for their analysis. A HPLC method for the analysis of 32P-postlabelled benzene-DNA adducts was developed and used to detect adducts formed from the reaction of DNA or individual deoxynucleotides with the metabolites para-benzoquinone (p-BQ) and hydroquinone (HQ). Reaction of DNA with BQ yielded four adducts, the major product being a deoxycytidine adduct. HQ formed a single detectable deoxyguanosine DNA adduct, which was a minor product of the reaction of DNA with p-BQ. The supF forward mutation assay was used to assess the mutagenicity of p-BQ and HQ after transfection of treated plasmid in the human kidney cell line, Ad293. Single base substitution mutations at GC base pairs (bp) predominated for each treatment. However, when the mutation spectra achieved for each treatment were compared they were shown to be significantly different (p=0.004). These results may suggest either a possible role for the minor benzene-deoxyguanosine adducts in benzene genotoxicity or that HQ is causing DNA modification via a different mechanism, such as oxidative damage.

Topics: Benzene; Benzoquinones; Cell Line; Chromatography, High Pressure Liquid; DNA Adducts; Genes, Suppressor; Humans; Hydroquinones; Mutagenicity Tests; Phosphorus Radioisotopes; RNA, Transfer

Reactive metabolites of benzene (BZ) play important roles in BZ-induced hematotoxicity. Although reactive metabolites of BZ covalently bind to DNA, the significance of DNA adduct formation in the mechanism of BZ toxicity is not clear. These studies investigated the covalent binding of the BZ metabolites hydroquinone(HQ) and 1,2,4-benzenetriol(BT) using the DNA [32P]postlabeling method and explored the potential relationship between DNA adduct formation and cell differentiation in human promyelocytic leukemia (HL-60) cells, a model system for studying hematopoiesis. Maturation of HL-60 cells to granulocytes, as assessed by light and electron microscopy, was significantly inhibited in cells that were pretreated with HQ or BT prior to inducing differentiation with retinoic acid (RA). The capacity of RA-induced cells to phagocytose sheep red blood cells (RBC) and to reduce nitroblue tetrazolium (NBT), two functional parameters characteristic of mature, differentiated neutrophils, was also inhibited in cells pretreated with HQ or BT. These BZ metabolite treatments induced DNA adduct formation in HQ- but not in BT-treated cells. These results indicate that whereas HQ and BT each block granulocytic differentiation in HL-60 cells, DNA adducts were observed only following HQ treatment. Thus DNA adduct formation may be important in HQ but not in BT toxicity.

Topics: Benzene Derivatives; Cell Death; Cell Differentiation; Cell Division; DNA; DNA Adducts; Granulocytes; HL-60 Cells; Humans; Hydroquinones; Leukemia, Myeloid; Mutagens; Phosphorus Radioisotopes; Tretinoin

We have examined DNA adduct formation and cytotoxicity in HL-60 cells treated with either hydroquinone (HQ) or p-benzoquinone (p-BQ). Treatment of HL-60 cells with either HQ or p-BQ produced the same DNA adduct. The DNA adduct level varied from 0.05 to 10 adducts per 10(7) nucleotides as a function of treatment time and concentration for both compounds. To achieve the same DNA adduct level required higher concentrations and longer treatment times with HQ compared to p-BQ. p-BQ was also more cytotoxic to HL-60 cells than HQ. Reaction of calf thymus DNA with a p-BQ/HQ mixture produced five adducts as detected by 32P-postlabeling. Two isomers of (hydroxy)-1,N2-benzetheno-2'- deoxyguanosine-3'-phosphate were isolated from the reaction of 2'-deoxyguanosine-3'-phosphate with a p-BQ/HQ mixture and one of the isomers was identified as adduct no. 1 of the DNA reaction. The DNA adduct formed in HL-60 cells treated with HQ or p-BQ did not correspond to any of the principal adducts formed in DNA reacted with p-BQ/HQ. This result suggests that cellular mechanisms modify DNA adduct formation by HQ and p-BQ.

Topics: Benzoquinones; Cell Survival; DNA; Humans; Hydroquinones; Leukemia, Promyelocytic, Acute; Phosphorus Radioisotopes; Tumor Cells, Cultured

The carcinogenicity of benzene has been considered to be in part mediated by its chemically reactive metabolic product benzoquinone (BQ), which is formed from the intermediary metabolites phenol and hydroquinone (HQ). We have evaluated the DNA-binding capability of these chemicals in vitro and in vivo by postlabeling. Treatment of rat Zymbal glands in culture with phenol and HQ or direct reaction of BQ with DNA produced DNA adducts, which were detectable by the nuclease P1-enhanced 32P-postlabeling assay as 5'-32P-labeled 3',5'-bisphosphate products. The enhancement of sensitivity in this assay is based on the previous finding that nuclease P1 hydrolyzes the phosphate attached to the 3' side of normal nucleotides but not the corresponding phosphate of most aromatic/bulky adducted nucleotides. Also based on this hydrolytic property of nuclease P1, we developed an additional sensitive procedure that permitted the detection of DNA lesions as 5'-32P-labeled products of dinucleotides, pXpN, or of nucleoside monophosphates, pX, where X and N indicate an adducted nucleoside and a normal nucleoside respectively. In the latter assay, adducted DNA was first digested with nuclease P1 and acid phosphatase to yield XpN and N. The latter were then 32P-labeled to yield [5'-32P] pXpN or 32P-labeled and treated with venom phosphodiesterase to obtain [5'-32P]pX. After optimization of enzymatic conditions, the modified nuclease P1 assay yielded adduct recoveries similar to those obtained by the bisphosphate assay for in vitro phenol-, HQ- and BQ-DNA adducts. Neither of the nuclease P1-enhanced postlabeling procedures showed exposure-specific adducts in vivo in the bone marrow, Zymbal gland, liver and spleen of female Sprague-Dawley rats at 24 h after the last of four single, daily p.o. doses of 75 mg/kg phenol or 150 mg/kg phenol/HQ (1:1). Our results show that phenol, HQ and BQ produce adducts in vitro, but corresponding adducts are not detected in vivo with phenol and phenol/HQ, even when measured by the standard and modified nuclease P1 postlabeling methods capable of detecting 1 adduct in 10(9-10) DNA bases.

Topics: Acid Phosphatase; Adenosine Triphosphate; Animals; Benzoquinones; DNA; Female; Hydroquinones; Nucleosides; Nucleotides; Phenol; Phenols; Phosphorus Radioisotopes; Pyrenes; Quinones; Rats; Rats, Inbred Strains; Single-Strand Specific DNA and RNA Endonucleases