Effect of age on the induction of 8-oxo-2'-deoxyguanosine-releasing enzyme in rat liver by gamma-ray irradiation.

Aged (27 months of age) and young (6 months of age) Fischer 344/DuCrj rats were exposed to gamma-ray irradiation, and their livers were compared for levels of oxidative DNA modifications and repair enzyme activities. The amounts of 8-oxo-2'-deoxyguanosine (8-oxodG) in the nuclear DNA of the livers of both young and aged rats increased immediately after irradiation, by 1.7-fold in the livers of young rats and 2.7-fold in the livers of the aged rats. Also, the rate of 8-oxodG decay was slower in the livers of the aged rats than in young rat liver, and remained above the baseline level even 1 week after irradiation. The activities of 8-oxodG-releasing enzymes peaked 2 and 6 h after irradiation in the livers of young and aged rats, respectively. The repair activity in the livers of the young rats was increased by sevenfold 2 h after irradiation, while the livers of the aged rats showed a twofold increase 6 h after irradiation. These results suggest that the ability to repair damaged DNA is lower in aged rats, and that the accumulation of oxidative DNA damage that takes place during aging may be related to this decline in repair activity.

Age-related changes in the induction of DNA polymerases in rat liver by gamma-ray irradiation.

DNA polymerase activities related to DNA repair were examined in the livers of young (6-month-old) and aged (27-month-old) rats irradiated with gamma-rays. The activity of DNA polymerase alpha was little changed in the livers of gamma-ray-irradiated rats, while DNA polymerases beta and gamma were induced in the livers of young and aged rats exposed by gamma-ray irradiation. These enzymes were induced from 2 to 6 h after irradiation of young and aged rats, respectively, although the induction in aged rats was weak. DNA polymerase beta activity in the livers of young rats irradiated with gamma-rays was 2-fold that in aged rats. Similarly, DNA polymerase gamma activity in the livers of young rats subjected to gamma-ray irradiation was 3-fold that in aged rats. The induction of proliferating cell nuclear antigen (PCNA) in the livers of aged rats irradiated with gamma-rays was also delayed compared with young rats. These results indicate that the decline in repair activity in aged rats leads to the accumulation of oxidative damage and DNA mutations in aged tissues.

The difference in the stimulation by putrescine of DNA synthesis using DNA polymerase extracts of normal rat liver or of tumour tissue or host liver from tumour-bearing rats.

Putrescine biosynthesis is elevated before DNA replication, and a stimulation of DNA synthesis by 20 mM putrescine has been found using an in vitro DNA synthesizing system. Furthermore, this stimulation of DNA synthesis by putrescine involves a particular factor (factor PA). This factor PA stimulates DNA polymerases alpha, beta, and gamma, and is present in nuclei and mitochondria but not in cytoplasm. Factor PA loses about 80% of its activity by heating at 45 degrees C for 15 min or by hydrolysis with 100 mg ml(-1) Enzygel trypsin. These properties indicate that factor PA is a protein. Its size is estimated to be about 2.1 S. DNA synthesis in nuclear and mitochondrial DNA polymerase extracts from tumour tissues and host livers of tumour-bearing rats are not stimulated by 20 mM putrescine. However, the addition of excess factor PA to DNA synthesizing systems using DNA polymerase extracts from proliferative tissues again results in a stimulation of DNA synthesis by exogenous putrescine. These findings indicate that the stimulatory effect of DNA synthesis in vitro by exogenous putrescine is controlled by the ratio between factor PA and endogenously synthesized putrescine in proliferative tissues or that sent by the bloodstream from proliferative tissues. These results suggest that a non-stimulatory effect of putrescine on DNA synthesis may be diagnostic in tumour-bearing patients.

Increase in DNA polymerase gamma in the hearts of adriamycin-administered rats.

It is hypothesized that the cause of myocardiopathy is oxidative damage to mitochondrial DNA. To clarify this hypothesis, DNA polymerase gamma activity, which is related to the final step of mitochondrial DNA repair or renewal, was measured. One cycle of treatment consisted of five injections of adriamycin over 5 days at a dose of 1 mg/kg of body weight per day and then 2 days resting time. DNA polymerase gamma activities in the heart after one cycle of treatment were lower than the control level. However, DNA polymerase gamma activities increased with continued adriamycin treatment, reaching a maximum level in the heart at 14 days after two cycles of adriamycin treatment. Induction of DNA polymerase gamma activity was found in rat heart following three and four cycles of administration. Under these conditions, it is doubtful that mitochondrial DNA is the direct target of adriamycin administration. The damaged mitochondrial DNA may be protected by actions of the renewal or repair systems, maintaining mitochondrial function in the heart. Rat hearts at 7 days after one cycle of adriamycin treatment show morphological changes in the mitochondria that include matrix swelling and cristae disorganization, as seen in cardiac cells by electron microscopy; however, 28 days after treatment, the mitochondria appear to have recovered.

SELECTIVE INHIBITION BY APHIDICOLIN OF THE ACTIVITY OF DNA POLYMERASE ALPHA LEADS TO BLOCKADE OF DNA SYNTHESIS AND CELL DIVISION IN SEA URCHIN EMBRYOS.

Aphidicolin at 2 µg/ml caused 90% inhibition of mitotic cell division of sea urchin embryos at the I-cell stage. However, at 40 µg/ml it did not affect meiotic maturational divisions of starfish oocytes, which do not involve DNA replication. At 2 µg/ml it caused 90% inhibition of incorporation of tritiated thymidine into DNA of sea urchin embryos but did not affect protein or RNA synthesis even at a higher concentration. At 2 µg/ml it also caused 90% inhibition of the activity of DNA polymerase α, obtained from the nuclear fraction of sea urchin embryos, but did not affect the activity of DNA polymerase ß or γ. These findings suggest that DNA polymerase α is responsible for replication of DNA in sea urchin embryos.