Carcinogen-induced DNA Damage Research Articles

Carcinogen induced DNA damage in isolated rat liver nuclei

Alkali-labile DNA lesions were detected by a hydroxylapatite batch assay following incubation of isolated rat liver nuclei with direct and indirectacting alkylating agents. N-Methyl- N′-nitro- N-nitrosoguanidine (MNNG) (20–200 μM) produced a steep, concentration-related decrease in double stranded DNA. In contrast, only minimal effects on DNA integrity were observed with methylmethane sulfonate (200 μM). The bifunctional alkylating agent, mechlorethamine produced an apparent increase in double-stranded DNA in accord with its ability to induce interstrand DNA crosslinks. The indirect-acting alkylating agent, dimethylnitrosamine (DMN) induced DNA damage only when an NADPH generating system was included in the incubation mixture. These results suggest that isolated rat liver nuclei may be a useful model for studying the effects of mutagenic and carcinogenic chemicals on the integrity of chromosomal DNA.

Acceptors for the poly ADP-ribosylation modification of chromatin structure are altered by carcinogen-induced DNA damage

Poly(ADP-Rib) polymerase is activated by strand breaks in DNA and appears to play an important role in DNA repair. The enzyme catalyses the poly ADP-ribosylation of histones and non-histone proteins, yet the contribution of these major alterations in chromatin composition have, as yet, not been critically evaluated with regard to DNA strand breaks. In the present study, the effects of N-methyl-N-nitrosourea (MNU) upon the poly ADP-ribosylation of nuclear protein acceptors have been identified and quantified at the oligonucleosomal level of chromatin. Treatment of HeLa cells with MNU (4.5 mM) for 1 h resulted in a reduction in the cellular NAD pool (30%), a 2-3 fold stimulation of poly ADP-ribosylation in isolated nuclei and in isolated oligonucleosomes. Of acceptors modified, the automodification of the polymerase was stimulated at least 3-fold. Analysis of the acid-soluble acceptors showed a stimulation in the modification of the core histones and a 2-fold increase in histone H1 poly ADP-ribosylation. This modification causes a novel crosslinking of the latter histone, and this has been studied as it relates to DNA strand breaks in the present work. In vivo treatment with MNU resulted in the synthesis of longer chain or more complex polymer species at the expense of the shorter chained ADP-ribose moieties.

12-O-tetradecanoyl-phorbol-13-acetate and its relationship to SCE induction in Syrian and Chinese hamster cells.

12-O-Tetradecanoyl-phorbol-13-acetate (TPA) in conditions that produce enhancement of ultraviolet light (UV) and x-irradiation Syrian hamster embryo cell (HEC) transformation did not cause further increase in the sister chromatid exchange (SCE) frequency induced by UV and x-irradiation, two physical carcinogens that differ in their mode of DNA interaction and efficiency of SCE induction. Several factors which might influence SCE induction by TPA were studied on HEC and Chinese hamster V79-4 cells. Heat-inactivated serum was used because of the possibility that a serum component may interfere with TPA ability to cause SCE. TPA effect on SCE was determined at the first and second division post treatment on cells exposed to different 5-bromodeoxyuridine (BrdUrd) concentrations. Independent of BrdUrd concentration (1-10 microgram/ml medium) and the number of cell divisions post treatment TPA (0.01-2 microgram/ml medium) was ineffective in inducing SCE in exponentially and stationary HEC cultures cultivated in medium supplemented with heat-inactivated serum. Also, TPA did not increase the SCE frequency in V79-4 Chinese hamster cells cultured in heat-inactivated or noninactivated serum. Although SCE induction, a cellular response to carcinogen-induced DNA damage, may be important for the induction of transformation by environmental agents, the enhancement of transformation frequency caused by TPA occurs without further DNA alterations involved in SCE formation.

Cellular responses to carcinogen-induced DNA damage and the role of DNA repair.

Cellular responses to carcinogen-induced DNA damage and the role of DNA repair.

Initiation of chemical carcinogenesis requires cell proliferation.

CELL proliferation has often been implicated in the development of cancer with chemicals1–3. Supportive evidence for this is the observation that several carcinogens that normally do not induce liver cancer in intact adult animals, especially with a single dose, become carcinogenic if administered as a single injection after partial hepatectomy (PH)1. In these conditions, it is thought that PH may act during initiation, presumably by fixation of some carcinogen-induced DNA damage through replication of the altered DNA. However, because carcinogenesis is a multi-step process, often involving the appearance of several new cell populations between the initial target cells and the ultimate cancer4,5, it is possible that the regenerative response of liver following PH could have a major effect on one or more steps subsequent to initiation. Thus, the use of a very late end-point (cancer) makes it difficult, if not impossible, to relate with any accuracy some very early event to any specific step in the carcinogenic process. Solt and Farber6 have recently developed a new approach to the sequential analysis of carcinogenesis in vivo that delineates the first few steps in the process and is a quantitative assay for initiation of liver cancer. We have used this model to investigate whether cell replication exerts its first effect on initiation or on some later step in the carcinogenic process. And if initiation is at least one site of action, when in the regenerative cell cycle is a carcinogen most effective and what biochemical events might be involved at this phase of the cell cycle?

A nonradioactive method for measuring DNA damage and its repair in nonproliferating tissues

The fluorometric assay of Kissane and Robins has been modified to monitor DNA in alkaline sucrose gradient fractions. Using this procedure the sedimentation analysis of DNA not only of liver, but also of brain, thymus, lung, pancreas, kidney, and skin was carried out. Like liver DNA, DNA released by the alkaline lysis of the above organs sedimented as heavy DNA (> 1 × 10 9 daltons). A good correspondence was obtained for the sedimentation profiles of liver DNA whether DNA in the gradient fractions was determined by the fluorometric method or by measuring radioactivity. Using the fluorometric assay, the strand breaks not only of liver DNA but also of brain and kidney DNA have been demonstrated following the intravenous administration of N-methylnitrosourea. Carcinogen-induced DNA damage and repair (as measured by sedimentation of DNA in alkaline sucrose gradients) in any organ including human biopsy specimens are potentially measurable by this procedure.

Studies on the mechanism of inhibition of 2-acetylaminofluorene toxicity by butylated hydroxytoluene

Male rats were placed on a diet containing 0.05% (w/w) of the hepatic carcinogen 2-acetylaminofluorene (AAF). They ceased to gain weight. However, when the carcinogenic diet was supplemented with butylated hydroxytoluene (BHT) (0.5% w/w), an antioxidant, the animals gained weight at approximately one-half the normal rate. This observation led to a series of experiments aimed at elucidating the mechanism(s) by which BHT reduced the toxicity of AAF. These initial studies were directed towards the effect of BHT on the extent and duration of the covalent binding of AAF with DNA. BHT feeding was shown to reduce the binding of carcinogen to hepatic DNA. Studies employing cells in culture demonstrated that BHT does not influence either excision repair or post-replication repair of DNA. These data indicate that a potential mechanism of action of BHT is at the anti-initiation level of carcinogen-induced DNA damage.