DNA adduction by phenol, hydroquinone, or benzoquinone in vitro but not in vivo: nuclease P1-enhanced ³²P-postlabeling of adducts as labeled nucleoside bisphosphates, dinucleotides and nucleoside monophosphates

Abstract

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.

The Toxicology of Hydroquinone — Relevance to Occupational and Environmental Exposure

“…112 In contrast to the clearly demonstrated formation of DNA adducts by HQ exposure in vitro, evidence for similar adduction in whole animal experiments is absent. Using two variations of the 32 P-postlabeling assay, Reddy et al 113 failed to demonstrate adducts in bone marrow, Zymbal gland, liver, or spleen DNA from female SD rats after HQ exposure (75 or 150 mg/kg/day, 4 days, p.o.). No increase in adducts by postlabeling was seen in kidney DNA from male or female F344 rats exposed to 50 mg/kg/day HQ, p.o., for 6 weeks.…”

Benzene and Leukemia

“…Lutz and Schlatter (1977) administered benzene by inhalation and suggested that with respect to covalent binding to hepatic DNA, benzene was among the poorest binding agents. Reddy et al (1990Reddy et al ( , 1994 treated rats with benzene by gavage for 5 days per week for up to 10 weeks at doses ranging from 200 to 500 mg/kg/day. At the end of 10 weeks they observed three faint spots in the post-abeling assay when he examined the Zymbal gland, but not in the marrow or other organs.…”

Benzene‐Associated Leukemia and its Risk Assessment

“…32P-postlabeling studies have demonstrated DNA adduct formation in the liver of New Zealand rabbits74) and in the bone marrow of B6C3Fi mice 75) treated with benzene ; however, Reddy et al [76][77][78] were unable to detect the formation of DNA adducts in various tissues of either Sprague-Dawley rats or B6C3Fi mice administered benzene or its hydroxylated metabolites.…”