32P-postlabeling assay for carcinogen-DNA adducts: nuclease P1-mediated enhancement of its sensitivity and applications.

Reddy, M. Vijayaraj; Randerath, Kurt

doi:10.1289/ehp.877641

Cited by 9 publications

(18 citation statements)

References 29 publications

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“…Polyethyleneimine (PEI)cellulose plates were prepared (9) in the laboratory. The sources of all other materials needed for 32P-postlabeling analysis of adducts have been documented previously (6)(7)(8).…”

Section: Methodsmentioning

confidence: 99%

“…An average yield of DNA from pooled Zymbal glands in several extractions corresponded to 17 (±6) ,ug DNA per rat. The presence of adducts in DNA was analyzed using a nuclease Pi-enhanced postlabeling assay (7,8). Briefly, DNA (10-15 dig) was enzymatically digested to 3'-deoxyribomonucleotides (12), which were then treated with nuclease Pi, which dephosphorylates nonnal nucleotides but not aromatic adducted nucleotides (8).…”

Section: Methodsmentioning

confidence: 99%

“…The sheet was soaked in water as before, dried, and developed in D4, perpendicular to the preceding development, in water to 1 cm, followed by 0.36 M sodium phosphate, 0.23 M Tris-HCl, pH 8.0, to the top, and dried. 32P-adducts were detected by screen-enhanced autoradiography (6,7). Adduct radioactivity was determined by excising the spots from the chromatograms and Cerenkov counting (6,7).…”

Section: Methodsmentioning

confidence: 99%

“…Adduct radioactivity was determined by excising the spots from the chromatograms and Cerenkov counting (6,7). An estimation of adduct levels, expressed as relative adduct labeling (RAL), was made from the values of adduct count rates, adjusted for background radioactivity, and the specific activity of y-32P-ATP determined by measuring the incorporation of 32p into a known amount of dAp (7,8). any test substance revealed that the glands at 6 hr (not shown) were histologically indistinguishable from those at 0 hr, i.e., the freshly removed tissue (Fig.…”

Section: Methodsmentioning

confidence: 99%

“…The capability of the cultured glands to metabolize benzene and its derivatives [catechol, 1,2,4-benzenetriol (BT), phenol, hydroquinone (HQ), and benzoquinone (BQ)], as well as 7,12-dimethylbenzanthracene (DMBA) and 2-acetylaminofluorene (AAF), was then assessed by measuring covalent binding of these compounds to DNA using a highly sensitive 32P-postlabeling assay (6,7). DNA adducts were readily detectable with all compounds tested, except benzene, which produced relatively low levels of adducts.…”

Section: Introductionmentioning

confidence: 99%

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A Method for In Vitro Culture of Rat Zymbal Gland: Use in Mechanistic Studies of Benzene Carcinogenesis in Combination with 32 P-Postlabeling

Reddy¹,

Blackburn²,

Irwin³

et al. 1989

Environmental Health Perspectives

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Zymbal glands were excised bilaterally from the ear ducts of female Sprague-Dawley rats (three/group), minced into approximately four fragments per gland, and transferred into a microtiter plate containing 1.5 mL per well of Waymouth's tissue culture medium supplemented with fetal calf serum, hydrocortisone, insulin, and gentamicin. After addition of a test compound or solvent vehicle, plates were incubated for 6, 24, 48, or 96 hr at 37°C in a humidified atmosphere of 5% CO2 in air. Tissue in culture for 6 hr was histologically indistinguishable from the freshly excised tissue, while that in culture for 24, 48, and 96 hr showed a progressive deterioration often with necrosis and/or squamous metaplasia. More pronounced deterioration was noted in samples treated with 750 or 1500 pg/mL of benzene. Using a nuclease Pi-enhanced 32P-postlabeling assay, aromatic DNA adducts were detected in cultured Zymbal glands exposed for 48 hr to benzene and its derivatives, as well as to 7,12-dimethylbenzanthracene (DMBA) and 2-acetylaminofluorene (AAF). Benzene produced very low levels of adducts (0.5 adducts per 109 nucleotides), whereas its congeners produced relatively high levels of adducts (50-2000 lesions per 109 nucleotides), which decreased in the order benzoquinone > hydroquinone > phenol > benzenetriol > catechol. Each adduct profile overall was characteristic for the compound studied, suggesting the formation of compound-specific electrophiles. AAF and DMBA adducts were identical to those formed in vivo in animals. Our results show that the Zymbal glands are capable of metabolizing different carcinogens to DNA-reactive intermediates, a process that may be causally associated with tumor formation in vivo in this organ.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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A Method for In Vitro Culture of Rat Zymbal Gland: Use in Mechanistic Studies of Benzene Carcinogenesis in Combination with 32 P-Postlabeling

Reddy¹,

Blackburn²,

Irwin³

et al. 1989

Environmental Health Perspectives

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Acidic cellular microenvironment modifies carcinogen-induced DNA damage and repair

et al. 2016

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Chronic inflammation creates an acidic microenvironment, which plays an important role in cancer development. To investigate how low pH changes the cellular response to the carcinogen benzo[a]pyrene (B[a]P), we incubated human pulmonary epithelial cells (A549 and BEAS-2B) with nontoxic doses of B[a]P using culturing media of various pH’s (extracellular pH (pHe) of 7.8, 7.0, 6.5, 6.0 and 5.5) for 6, 24 and 48 h. In most incubations (pHe 7.0–6.5), the pH in the medium returned to the physiological pH 7.8 after 48 h, but at the lowest pH (pHe < 6.0), this recovery was incomplete. Similar changes were observed for the intracellular pH (pHi). We observed that acidic conditions delayed B[a]P metabolism and at t = 48 h, and the concentration of unmetabolized extracellular B[a]P and B[a]P-7,8-diol was significantly higher in acidic samples than under normal physiological conditions (pHe 7.8) for both cell lines. Cytochrome P450 (CYP1A1/CYP1B1) expression and its activity (ethoxyresorufin-O-deethylase activity) were repressed at low pHe after 6 and 24 h, but were significantly higher at t = 48 h. In addition, a DNA repair assay showed that the incision activity was ~80% inhibited for 6 h at low pHe and concomitant exposure to B[a]P. However, at t = 48 h, the incision activity recovered to more than 100% of the initial activity observed at neutral pHe. After 48 h, higher B[a]P-DNA adduct levels and γ-H2AX foci were observed at low pH samples than at pHe 7.8. In conclusion, acidic pH delayed the metabolism of B[a]P and inhibited DNA repair, ultimately leading to increased B[a]P-induced DNA damage.Electronic supplementary materialThe online version of this article (doi:10.1007/s00204-016-1907-4) contains supplementary material, which is available to authorized users.

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Polycyclic Aromatic Hydrocarbon-DNA Adducts in Prostate Cancer

Rybicki¹,

Rundle²,

Savera

et al. 2004

Cancer Research

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The formation of DNA adducts can lead to DNA replication errors and the potential for carcinogenesis. DNA adducts have been detected in prostate cells, but the distribution of adducts with respect to prostate cancer risk factors and histology is unknown. In a study of 130 Caucasian (n ‫؍‬ 61) and African-American (n ‫؍‬ 69) men with prostate cancer who underwent radical prostatectomy, we quantified polycyclic aromatic hydrocarbon (PAH)-DNA adducts in prostate tumor and adjacent nontumor cells by immunohistochemistry. A strong correlation between paired adduct levels in the two cell types was observed (r ‫؍‬ 0.56; P < 0.0001); however, nontumor cells had a significantly higher level of adducts compared with tumor (0.30 absorbance units ؎ 0.05 versus 0.17 absorbance units ؎ 0.04; P < 0.0001). Variables significantly associated with PAH-DNA adduct levels in tumor cells included primary Gleason grade, tumor volume, and log-transformed prostate-specific antigen (PSA) at time of diagnosis. Tumors with a primary Gleason grade of 5 had significantly lower PAH-DNA adduct levels than tumor cells with a primary Gleason grade of 3 or 4 (P < 0.0001 for both). Tumors that involved 10% or less of the prostate gland had significantly higher PAH-DNA adduct levels than tumors that involved 15 to 20% of the prostate gland (P ‫؍‬ 0.004). PSA levels were inversely associated with PAH-DNA adduct levels in tumor cells (P ‫؍‬ 0.009). A similar, albeit less significant, inverse association was observed between PSA and PAH-DNA adduct levels in nontumor cells (P ‫؍‬ 0.07). Interestingly, increasing primary Gleason grade was associated with increasing PAH-DNA adduct levels in adjacent nontumor cells (P ‫؍‬ 0.008). Our results show that PAH-DNA adducts are present in the prostate but vary with regard to cellular histology. In prostate tumor cells, decreased cellular differentiation and increased tumor proliferation may reduce PAH-DNA adduct levels.

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32P-postlabeling assay for carcinogen-DNA adducts: nuclease P1-mediated enhancement of its sensitivity and applications.

Cited by 9 publications

References 29 publications

A Method for In Vitro Culture of Rat Zymbal Gland: Use in Mechanistic Studies of Benzene Carcinogenesis in Combination with 32 P-Postlabeling

A Method for In Vitro Culture of Rat Zymbal Gland: Use in Mechanistic Studies of Benzene Carcinogenesis in Combination with 32 P-Postlabeling

Acidic cellular microenvironment modifies carcinogen-induced DNA damage and repair

Polycyclic Aromatic Hydrocarbon-DNA Adducts in Prostate Cancer

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