The binding of 7,12-dimethylbenz(a)anthracene to replicating and non-replicating DNA in cell culture

Yuspa, Stuart H.; Eaton, Sol del Ande; Morgan, David L.; Bates, Richard

doi:10.1016/0009-2797(69)90009-x

Cited by 21 publications

(4 citation statements)

References 12 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Unfortunately, the significant binding site for transformation in terms of polycyclic hydrocarbon carcinogens is unknown. For example, dimethylbenz(a)anthracene is known to bind well to I)NA, regardless of the rate of DNA synthesis (11), and to bind nonreplicating DNA as well as to replicating DNA (12).…”

Section: Discussionmentioning

confidence: 99%

X-Irradiation Enhancement of Transformation by Benzo(a)Pyrene in Hamster Embryo Cells

DrPaolo

Donovan

Nelson

1971

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

The addition of benzo(a)pyrene to cells obtained from Syrian hamster embryo cultures that had been plated to produce discrete colonies resulted in transformation. Altered colonies were not seen in controls. Prior irradiation of hamster embryo cells increased the transformation by benzo(a)pyrene, both per cell and per total colony. Maximum enhancement occurred when benzo(a)pyrene was added 48 hr after irradiation. Enhancement was greatest after 250-500 R, and decreased with higher doses of radiation. Although the link between radiation (both x-and UVirradiation) and cancer has been recognized since the discoveries of x-rays and natural radioactivity, the use of physical agents as carcinogens or cocarcinogens for in vitro studies has been extremely limited. X-irradiation has been shown to promote in vitro transformation in combination with polyoma (4) or simian virus 40 (5, 6) in hamster embryonic secondary cultures, hamster cell line BHK 21/13, and 3T3 mouse cell lines. Evidence was presented that the effect in established cell lines was not due to selection of preexisting cells with a greater susceptibility to transformation. Cell transformation in vitro by x-irradiation only has been reported in primary cell lines of hamster embryo cells (7), but has not been observed in the irradiated controls of the experiments involving radiation and viruses or after repeated weekly irradiation of a hypotetraploid fibroblast line derived from mouse spleen (8).Abbreviation: BP, benzo(a)pyrene. MATERIALS AND METHODSThe tissue-culture fluids and medium as well as the feeder layer cultures, the source and culture of cells from minced hamster embryos, and the standard conditions for the transformation assay have been described in detail (2). Cells derived from whole rat or hamster fetuses were grown as monolayers in Dulbecco's modification of Eagle's medium (MEM) with either 10% calf serum or 10% fetal bovine serum. In all experiments 2-to 4-day-old secondary or tertiary hamster cultures obtained by seeding 5 X 106 cells/100 ml plastic dish were used. Feeder layers composed of secondary cultures of rat embryo cells were irradiated as confluent monolayer cultures in 2 ml of medium. A Picker Portable Industrial x-ray apparatus (T55-433) was used at 110 kV and 5 mA, 2225 R/min, adjusted so that a total of 5000 R in air was delivered to a distance of 22.2 cm. Hamster cells were irradiated as confluent monolayers in 100-mm plastic Petri dishes with a Westinghouse Quandrocondex machine with two tubes, above and below, with the following factors: 200 kV (CP), 0.25 Cu + 0.55 mm Al filtration in each tube (HVL 0.9 mm Cu), 15 mA, 54 cm TSD, output 139 R/min. After being irradiated with 150-1000 R, the cells were plated on irradiated rat fibroblast feeder layers in 60-mm Petri dishes. Each value represents data obtained from at least 12 plates. Because unirradiated and irradiated cells had different survival rates, it was necessary to vary the number of hamster cells seeded to obtain the same approximate number of colonies. At desig...

show abstract

Section: Discussionmentioning

confidence: 99%

X-Irradiation Enhancement of Transformation by Benzo(a)Pyrene in Hamster Embryo Cells

DrPaolo

Donovan

Nelson

1971

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

show abstract

“…The results show that this poor binding is not due to a requirement to bind to DNA which is undergoing replication. Although the binding is somewhat higher to this newly synthesized peak, it is still extremely poor compared to that shown for the strong carcinogen DMBA (1) even though the latter was used at a much lower concentration (Table I).…”

Section: (2)mentioning

confidence: 88%

“…Previous studies (1) have demonstrated that binding of 7, 12-dimethylbenz [ a ] anthracene (DMBA) to DNA does not require DNA sy,nthesis. DNA which had replicated in the presence of DMBA was separated from nonreplicating DNA by the addition of 5-bromodeoxyuridine (BUDR) to cultures of fetal mouse skin and subsequent isopycnic density sedimentation of extracted DNA.…”

mentioning

confidence: 99%

See 1 more Smart Citation

The Binding of Benz [a] anthracene to Replicating and NonReplicating DNA in Cell Culture

Yuspa

Bates

1970

Experimental Biology and Medicine

View full text Add to dashboard Cite

Previous studies (1) have demonstrated that binding of 7, 12-dimethylbenz [ a ] anthracene (DMBA) to DNA does not require DNA sy,nthesis. DNA which had replicated in the presence of DMBA was separated from nonreplicating DNA by the addition of 5-bromodeoxyuridine (BUDR) to cultures of fetal mouse skin and subsequent isopycnic density sedimentation of extracted DNA. More binding was found to DNA which had not replicated while DMBA was present than to that which had. That this was the case for the strong carcinogen DMBA did not necessarily imply that all the polycyclic hydrocarbons acted this way with regards to binding during the cell cycle. In fact, Alfred and DiPaolo ( 2 ) had reported that the weakly carcinogenic hydrocarbon benz [ a ] anthracene (BA) (3-5) reached a maximum binding to hamster embryo cell DNA during the first 4 hr of the release of the cells from a thymidine block. This was the same time that maximum DNA synthesis had occurred in their culture system. DMBA in their system did not reach a maximum binding until 24 hr, a time when more than one complete cycle had occurred. Furthermore, weakly carcinogenic hydrocarbons had previously been shown to be very poor binders to DNA when painted on mouse skin. (6) These studies suggested the possibility that weak carcinogens could only bind to DNA that was being replicated. If this were so, it might indicate that binding of carcinogens to DNA during replication was not as important to carcinogenesis as binding in some other part of the cell cycle, if indeed binding was important. For this reason BA was tested in the cell cultures system which utilized BUDR to separate the replicating from nonreplicating DNA. Materials and Methods. The cell culture technique, basic media and DNA isolation were performed as previously reported ( 1). The preparation of the hydrocarboncontaining medium and the scintillation counting procedure are described below.BA Medium. 3H-BA generalIy labeled was obtained from Amersham-Searle at a specific activity of 750 mCi/mmole. Approximately 5000 pCi (1500 pg) in 5 ml of benzene was exposed to a stream of nitrogen until the benzene had evaporated. The 3H-BA was redissolved in 100 ml of acetone in semidarkness and slowly added to 15 ml of fetal calf serum being slowly stirred in an ice bath. This was allowed to mix for 30 min in the dark and an additional 100 pl of acetone was used to rinse the 3H-BA ampoule and then added slowly to the serum. The solution was mixed for 4 hr in the dark in an ice bath and then added to 85 ml of thyminefree medium 199 and 1 ml of antibiotic mixture giving a final radioactivity of 50 pCi of 3H-BA per Jml 3H-BA/ml (1 5 pg/ml) . This stock solution was stored in the dark at 4 O . For the experimental medium a 1 : 20 dilution of the stock mixture was used with either BUDR or thymine-containing medium making a final radioactivity of 2.5 pCi/ml (0.75/ Collection of fractions and scintillation counting. Following ultracentrifugation of the isolated DNA a t 45,000 rpm for 66 hr, 0.4-ml fractions were collecte...

show abstract

Chemically and Metabolically Induced DNA Adducts: Relationship to Chemical Carcinogenesis

Heidelberger¹

1976

Aging, Carcinogenesis, and Radiation Biology

View full text Add to dashboard Cite

The binding of 7,12-dimethylbenz(a)anthracene to replicating and non-replicating DNA in cell culture

Cited by 21 publications

References 12 publications

X-Irradiation Enhancement of Transformation by Benzo(a)Pyrene in Hamster Embryo Cells

X-Irradiation Enhancement of Transformation by Benzo(a)Pyrene in Hamster Embryo Cells

The Binding of Benz [a] anthracene to Replicating and NonReplicating DNA in Cell Culture

Chemically and Metabolically Induced DNA Adducts: Relationship to Chemical Carcinogenesis

Contact Info

Product

Resources

About