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Replicative repair of UV damage to normal human cells
335 1 K. Aoki, Y. Taguchi, H. Matsudaira and T. Ishikawa *, National Institute of Radiological Sciences, Chiba, and * Cancer Institute, Tokyo (Japan)
Hepatic changes of the teleost, Oryzias latipes after treatment with AF-2 and nitrofurazone Chronic effects of 2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide (AF-2) and nitrofurazone were examined in a teleost, Oryzias latipes. Groups of adult fish (1 year old) were maintained in well water containing AF-2 (2--5 pg/ml) and nitrofurazone (5--30 pg/ml) in the dark for 30 days at 25°C, then transferred to well water not containing the drugs and kept in the light. Survival of the fish five months after the transfer was almost the same as that of the untreated control. A marked change was observed in the liver of the fish subjected to the treatment, particularly from 2 or 3 months after the transfer. The histological features of the change did not vary in groups treated with AF-2 and nitrofurazone. One of the most striking changes in the liver was the development of multiple oval degenerative foci. The largest were less than 1 mm in diameter, and they were sharply defined from surrounding liver tissue, but not encapsulated. These lesions consisted of degenerating hepatic cells with markedly swollen, lightly stained cytoplasm and small pyknotic nuclei. Large acellular lacunae filled with amorphous material were also noted. Even when the treatment was suspended these lesions seemed to remain for 4--5 months. Complete serial sectioning usually revealed direct penetration of extravasated erythrocytes from moderate sized vessels into these degenerative foci. Surrounding hepatic cells had markedly basophilic, hypertrophied cytoplasm in treated groups, but the original hepatic structure was retained. No neoplastic change was found.
2 Y. Fujiwara and M. Tatsumi, Department of Radiation Biophysics, Kobe University School of Medicine, Kobe (Japan) Replicative repair of UV damage to normal human cells Caffeine-sensitive post-replication repair has been fairly well documented in UV-irradiated xeroderma pigrnentosum (XP) cells from rodents and humans. Here we show that normal human cells perform an alternative replicative repair of UV lesions. Smaller nascent segments made in cells from normal human skin (NHSF) shortly after UV exposure were almost normally elongated during chase in caffeine (2 mM), as revealed by alkaline sucrose gradient. Unlike the effect in Agroup XP cells, the mean lethal UV dose for NHSF cells was not reduced by caffeine. The DNA from NHSF cells that had been incubated in unlabeled BUdR for the first 2 h after exposure to 20 J m -~ and then 2 h in [3H]TdR was banded in alkaline CsCl: [3H]-radioactivity was totally associated with DNA banded at a normal density of 1.725 g m1-1, but not with heavy DNA, showing that de
336 novo gap-filling repair may not take place in the cells. Further, NHSF cells were pre-labeled with B U d R for 2 h, irradiated with 0 or 20 J m -2, and then incubated in [3H]BUdR for 1 to 2 h. The neutral CsC1 profile of the extracted DNA s h o w ed that the DNA replicating in irradiated cells was composed of a large portion of intermediate-density DNA (1.730 g m1-1 for hybrid 1.752 g m1-1 and light band 1.700 g ml-'), hybrid (usually seen in unirradiated cells) and a small amount of heavy-heavy DNA (1.805 g ml-1). The intermediate and heavyheavy DNAs were chased into a single hybrid peak after an additional 2-h chase in BUdR. Thus, replication of NHSF cells seems blocked at a UV lesion without forming a gap, and perhaps a newly synthesized strand may be utilized as a template to replicate a damaged site in the parental DNA, thereby restoring the proper genetic information. A Grant-in-Aid from the Ministry of Education is acknowledged. A b b r e v i a t i o n s : TdR, thymidine; BUdR, 5-bromodeoxyuridine.
3 S. Hitotsumachi and Y. Kikuchi, Drug Safety Research Centre, Takeda Chemical Industries Ltd., Osaka {Japan) Mitomycin-C-induced dominant lethality in mice The dose effect of mitomycin C (MC) in the induction of dominant lethality was studied in CF1 mice. Adult males were given a single i.p. injection of 1, 2 or 3 mg of MC per kg. The treated male was mated with untreated virgin females during 7 weeks after treatment. On the 13th day of pregnancy the female was killed and examined for corpora lutea as well as living and dead implants. The treatment at 3 mg/kg induced marked pre- and post-implantation dominant lethality J=l
number of living embryos per female in experiment number of living embryos per female in control
The sensitive stage was from spermatogonia to early spermatids. At 2 mg/kg, only spermatocytes were sensitive in regard to increase of J value. No increase of J value was induced at any stage of spermatogenesis with MC at 1 mg/kg. Increase of dominant lethality in the present study was always associated with high pre-implantation egg loss, which could be due to decrease of sperm in the ejaculate [Kratochvilova, Mutation Res., 21 (1973) 192]. To examine this possibility, post-implantation dominant lethality J ' = 1 -- number of living embryos/all implants in experiment number of living embryos/all implants in control was also calculated. According to this J ' index, 3 mg of MC per kg seemed to have induced the mutation only in late spermatocytes and early spermatids. However, the J ' index was increased, though slightly, even with MC at 1 mg/kg at the late spermatocyte stage.
Hepatic changes of the teleost, Oryzias latipes after treatment with AF-2 and nitrofurazone Chronic effects of 2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide (AF-2) and nitrofurazone were examined in a teleost, Oryzias latipes. Groups of adult fish (1 year old) were maintained in well water containing AF-2 (2--5 pg/ml) and nitrofurazone (5--30 pg/ml) in the dark for 30 days at 25°C, then transferred to well water not containing the drugs and kept in the light. Survival of the fish five months after the transfer was almost the same as that of the untreated control. A marked change was observed in the liver of the fish subjected to the treatment, particularly from 2 or 3 months after the transfer. The histological features of the change did not vary in groups treated with AF-2 and nitrofurazone. One of the most striking changes in the liver was the development of multiple oval degenerative foci. The largest were less than 1 mm in diameter, and they were sharply defined from surrounding liver tissue, but not encapsulated. These lesions consisted of degenerating hepatic cells with markedly swollen, lightly stained cytoplasm and small pyknotic nuclei. Large acellular lacunae filled with amorphous material were also noted. Even when the treatment was suspended these lesions seemed to remain for 4--5 months. Complete serial sectioning usually revealed direct penetration of extravasated erythrocytes from moderate sized vessels into these degenerative foci. Surrounding hepatic cells had markedly basophilic, hypertrophied cytoplasm in treated groups, but the original hepatic structure was retained. No neoplastic change was found.
2 Y. Fujiwara and M. Tatsumi, Department of Radiation Biophysics, Kobe University School of Medicine, Kobe (Japan) Replicative repair of UV damage to normal human cells Caffeine-sensitive post-replication repair has been fairly well documented in UV-irradiated xeroderma pigrnentosum (XP) cells from rodents and humans. Here we show that normal human cells perform an alternative replicative repair of UV lesions. Smaller nascent segments made in cells from normal human skin (NHSF) shortly after UV exposure were almost normally elongated during chase in caffeine (2 mM), as revealed by alkaline sucrose gradient. Unlike the effect in Agroup XP cells, the mean lethal UV dose for NHSF cells was not reduced by caffeine. The DNA from NHSF cells that had been incubated in unlabeled BUdR for the first 2 h after exposure to 20 J m -~ and then 2 h in [3H]TdR was banded in alkaline CsCl: [3H]-radioactivity was totally associated with DNA banded at a normal density of 1.725 g m1-1, but not with heavy DNA, showing that de
336 novo gap-filling repair may not take place in the cells. Further, NHSF cells were pre-labeled with B U d R for 2 h, irradiated with 0 or 20 J m -2, and then incubated in [3H]BUdR for 1 to 2 h. The neutral CsC1 profile of the extracted DNA s h o w ed that the DNA replicating in irradiated cells was composed of a large portion of intermediate-density DNA (1.730 g m1-1 for hybrid 1.752 g m1-1 and light band 1.700 g ml-'), hybrid (usually seen in unirradiated cells) and a small amount of heavy-heavy DNA (1.805 g ml-1). The intermediate and heavyheavy DNAs were chased into a single hybrid peak after an additional 2-h chase in BUdR. Thus, replication of NHSF cells seems blocked at a UV lesion without forming a gap, and perhaps a newly synthesized strand may be utilized as a template to replicate a damaged site in the parental DNA, thereby restoring the proper genetic information. A Grant-in-Aid from the Ministry of Education is acknowledged. A b b r e v i a t i o n s : TdR, thymidine; BUdR, 5-bromodeoxyuridine.
3 S. Hitotsumachi and Y. Kikuchi, Drug Safety Research Centre, Takeda Chemical Industries Ltd., Osaka {Japan) Mitomycin-C-induced dominant lethality in mice The dose effect of mitomycin C (MC) in the induction of dominant lethality was studied in CF1 mice. Adult males were given a single i.p. injection of 1, 2 or 3 mg of MC per kg. The treated male was mated with untreated virgin females during 7 weeks after treatment. On the 13th day of pregnancy the female was killed and examined for corpora lutea as well as living and dead implants. The treatment at 3 mg/kg induced marked pre- and post-implantation dominant lethality J=l
number of living embryos per female in experiment number of living embryos per female in control
The sensitive stage was from spermatogonia to early spermatids. At 2 mg/kg, only spermatocytes were sensitive in regard to increase of J value. No increase of J value was induced at any stage of spermatogenesis with MC at 1 mg/kg. Increase of dominant lethality in the present study was always associated with high pre-implantation egg loss, which could be due to decrease of sperm in the ejaculate [Kratochvilova, Mutation Res., 21 (1973) 192]. To examine this possibility, post-implantation dominant lethality J ' = 1 -- number of living embryos/all implants in experiment number of living embryos/all implants in control was also calculated. According to this J ' index, 3 mg of MC per kg seemed to have induced the mutation only in late spermatocytes and early spermatids. However, the J ' index was increased, though slightly, even with MC at 1 mg/kg at the late spermatocyte stage.