The combined action of chemical carcinogens on DNA repair in human cells

Summary Excision repair was studied in normal human and ataxia telangiectasia (AT) cells proficient in repair of UV and its mimetic chemicals, and in xeroderma pigmentosum group C (XP C) cells (deficient in repair of UV and its mimetics), after treatment with several combinations of chemical carcinogens, by the photolysis of bromodeoxyuridine incorporated into parental DNA during repair. Results indicate that repair was additive in AT, and XP C cells treated with N-acetoxy-2-acetylaminofluorene (AAAF) plus ethyl methanesulfonate (EMS) or methyl methanesulfonate (MMS) indicating that there are different rate limiting steps for removal of both types of damage. Data on the combinations of 4-nitroquinoline 1-oxide (4NQO) plus MMS or EMS are difficult to interpret, but they do not indicate inhibition of DNA repair.Research carried out under the auspices of the U.S. Dept. of Energy     

Poly(ADP-ribose) polymerase activity prevents signaling pathways for cell cycle arrest after DNA methylating agent exposure

Mouse fibroblasts, deficient in DNA polymerase beta, are hypersensitive to monofunctional DNA methylating agents such as methyl methanesulfonate (MMS). Both wild-type and, in particular, repair-deficient DNA polymerase beta null cells are highly sensitized to the cytotoxic effects of MMS by 4-amino-1,8-naphthalimide (4-AN), an inhibitor of poly(ADP-ribose) polymerase (PARP) activity. Experiments with synchronized cells suggest that exposure during S-phase of the cell cycle is required for the 4-AN effect. 4-AN elicits a similar extreme sensitization to the thymidine analog, 5-hydroxymethyl-2'-deoxyuridine, implicating the requirement for an intermediate of DNA repair. In PARP-1-expressing fibroblasts treated with a combination of MMS and 4-AN, a complete inhibition of DNA synthesis is apparent after 4 h, and by 24 h, all cells are arrested in S-phase of the cell cycle. Continuous incubation with 4-AN is required to maintain the cell cycle arrest. Caffeine, an inhibitor of the upstream checkpoint kinases ATM (ataxia telangiectasia-mutated) and ATR (ATM and Rad3-related), has no effect on the early inhibition of DNA synthesis, but cells are no longer able to maintain the block after 8 h. Instead, the addition of caffeine leads to arrest of cells in G(2)/M rather than S-phase after 24 h. Analysis of signaling pathways in cell extracts reveals an activation of Chk1 after treatment with MMS and 4-AN, which can be suppressed by caffeine. Our results suggest that inhibition of PARP activity results in sensitization to MMS through maintenance of an ATR and Chk1-dependent S-phase checkpoint.     

Increased PARP-1 association with DNA in alkylation damaged, PARP-inhibited mouse fibroblasts

Treatment of base excision repair-proficient mouse fibroblasts with the DNA alkylating agent methyl methanesulfonate (MMS) and a small molecule inhibitor of PARP-1 results in a striking cell killing phenotype, as previously reported. Earlier studies showed that the mechanism of cell death is apoptosis and requires DNA replication, expression of PARP-1, and an intact S-phase checkpoint cell signaling system. It is proposed that activity-inhibited PARP-1 becomes immobilized at DNA repair intermediates, and that this blocks DNA repair and interferes with DNA replication, eventually promoting an S-phase checkpoint and G(2)-M block. Here we report studies designed to evaluate the prediction that inhibited PARP-1 remains DNA associated in cells undergoing repair of alkylation-induced damage. Using chromatin immunoprecipitation with anti-PARP-1 antibody and qPCR for DNA quantification, a higher level of DNA was found associated with PARP-1 in cells treated with MMS plus PARP inhibitor than in cells without inhibitor treatment. These results have implications for explaining the extreme hypersensitivity phenotype after combination treatment with MMS and a PARP inhibitor.     

Repair Replication and Photorepair of DNA in Larvae of DROSOPHILA MELANOGASTER

Repair replication of DNA has been studied in first instar larvae of Drosophila melanogaster with isopycnic centrifugation techniques. Larvae were fed BUdR, FUdR, streptomycin, penicillin, and Fungazone for two to four hours prior to exposure to UV, X-rays, MMS, or EMS. Feeding was continued for four hours in the presence of (3)HBUdR and DNA was isolated from whole larvae. Repair replication is stimulated by each of these agents. MMS is about 10 times as potent as EMS in stimulating repair synthesis. A dose of 200 ergs/mm(2) largely saturates the level of repair replication observed after UV irradiation. Repair replication rises between 0 and 80,000 R of X-rays before falling off. Semiconservative synthesis is seriously inhibited above a dose of 40,000 R of X-rays. Photorepair has been detected as a reduction in repair synthesis resulting from post-irradiation exposure to photoreactivating light. The same treatment has no detectable effect on X-ray-stimulated repair replication. Repair replication is insensitive to the presence of caffeine or hydroxyurea during the final incubation, although semiconservative synthesis is strongly inhibited by these agents. A mixture of BUdR and (3)HTdR can be used to replace (3)HBUdR in detecting repair replication.     

Comparative analysis of caffeine and 3-aminobenzamide as DNA repair inhibitors in Syrian baby hamster kidney cells

The effects of caffeine and 3-aminobenzamide (3-AB) on Syrian baby hamster kidney cells treated with DNA-alkylating agents and ultraviolet-light suggest that two different DNA-repair mechanisms are involved. Both these agents enhanced the cell kill after methyl methanesulfonate (MMS) treatment. However, enhanced lethality was observed only with caffeine post-treatment when cells were exposed to nitrogen mustard (HN2) or ultraviolet light (UV); 3-AB did not appreciably change cell killing by these agents. With MMS-treated cultures, the effect of caffeine was maximal about 16 h later. The effect of 3-AB on the other hand, was exerted during the first 4 h after exposure to MMS. Caffeine's effect on cell survival could be abolished by low concentrations of cycloheximide, whereas 3-AB's effect could not. Furthermore, the G2 block in cell cycle progression, after MMS treatment, was not observed if the cells were post-treated with caffeine. In the presence of 3-AB, MMS-treated cells were arrested in G2 phase at a much earlier time compared to cells not treated with 3-AB. Finally caffeine post-treatment produced a 10-fold increase in nuclear fragmentation in MMS-treated cells. 3-AB did not cause nuclear fragmentation by itself but further enhanced the nuclear fragmenting effect of caffeine when both agents were present during the posttreatment. Therefore, we propose that 3-AB and caffeine each prevent a different repair mechanism from being effective.     

The relevance of caffeine post-treatment to SCE incidence induced in Chinese hamster cells

The influence of caffeine post-treatment on sister-chromatid exchanges (SCE) and chromosomal aberration frequencies on Chinese hamster cells exposed to a variety of chemical and physical agents followed by bromodeoxyuridine (BrdUrd) was determined. After 2 h treatment, N-methyl-N′-nitrosoguanidine (MNNG) and cis-platinum(II)diamine dichloride (cis-Pt(II)) induced a 7- and 6-fold increase in SCE, respectively, while 4-nitroquinoline-1-oxide (4NQO), methyl methanesulfonate (MMS), proflavine, and N-hydroxyfluorenylacetamide (OH-AAF) caused a 2–3-fold increase in SCE compared to controls treated with BrdUrd alone. Ultraviolet light doubled the number of SCE. The lowest increase of SCE was obtained with bleomycin and X-irradiation. Caffeine post-treatment caused a statistically significant increase in the frequency of SCE induced by UV- and X-irradiation as well as by 4NQO and MMS but did not alter the number of SCE induced by MNNG, cis-Pt(II), proflavine, OH-AAF, and bleomycin.

Caffeine post-treatment increased the number of cells with chromosomal aberrations induced by MNNG, cis-Pt(II), UV, 4NQO, MMS, and proflavine. With the exception of proflavine, these agents are dependent on DNA and chromosome replication for the expression of the chromosomal aberrations. Caffeine enhancement of cis-Pt(II) chromosomal aberrations occurred independently of the time interval between treatment and chromosome preparations. Chromosomal damage produced by bleomycin and X-irradiation, agents known to induce chromosomal aberrations independent of “S” phase of the cell cycle, as well as the damage induced with OH-AAF was not influenced by caffeine post-treatment.

The enhancement by caffeine, an inhibitor of the gap-filling process in post-replication repair, of chromosomal aberrations induced by “S” dependent agents, is consistent with the involvement of this type of repair in chromosomal aberration formation. The lack of inhibition of SCE frequency by caffeine indicates that post-replication repair is probably not important in SCE formation.     

Choloroquine inhibition of repair of DNA damage induced in mammalian cells by methyl methanesulfonate

Chloroquine (ClQ) inhibited the repair of DNA damage produced in cultured rat liver cells by methyl methanesulfonate (MMS). MMS caused fragmentation of single-strand DNA in alkaline sucrose gradients. Repair of the damage was followed by observing the restoration of the normal sedimentation pattern at intervals after treatment. Repair was significant by 7 h and nearly complete at 24 h. Addition of ClQ during the repair peiod markedly reduced the rate of repair. Also, ClQ increased the lethality of MMS, which could be due to the inhibition of repair. ClQ was found to inhibit protein synthesis, but the effect on repair is probably not due entirely to this action since comparable inhibition of protein synthesis by cycloheximide produced a lesser degree of delay in repair.     

Unscheduled DNA synthesis induced in mouse spermatids after combined treatment with methyl methanesulfonate and X-rays.

Unscheduled DNA synthesis (UDS), which is considered to be DNA repair, has been studied in early- to mid-spermatid stages of the mouse after combined treatments with X-rays and methyl methanesulfonate (MMS). UDS in spermatids was detected by giving testicular injections of [methyl-3H]thymidine ([3H]dThd) and making use of the fact that no scheduled DNA synthesis occurs in the germ cells after the last S period in primary spermatocytes. X-rays and MMS are each able to induce UDS in mouse spermatids. However, there was a statistically significant reduction in the amount of UDS observed when X-ray exposures of from 200 to 600 R were given 4 h before an i.p. injection of 75 mg/kg of MMS and concurrent testicular injections of [3H]dThd. This reduction in UDS is more than can be explained by the completion of repair of X-ray-induced DNA lesions. We suggest that the reduction in UDS is the result of an X-ray-produced impairment of a least a part of the repair mechanism involved in correcting MMS-induced DNA lesions. When the time interval between a 600-R X-ray exposure and MMS treatment was between 3 and 20 h (latest time interval s;udied) there was a statistically significant reduction of UDS in the spermatids. No significant decrease in UDS response occurred when the time interval between radiation exposure and MMS treatment was less than approximately 3 h.     

Cytotoxicity of monofunctional alkylating agents methyl methanesulfonate and methyl-N′-nitro-N-nitrosoguanidine have different mechanisms of toxicity for 10T1/2 cells

N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) and methyl methanesulfonate (MMS) are directly active alkylating agents that methylate cellular macromolecules by SN₁ and SN₂ mechanisms, respectively. These two chemicals produce similar types of alkylation products in DNA and a similar level of total alkylations on a molar basis, but strikingly different proportions of alkylations of ring oxygen atoms of purines and pyrimidines. Because of this attribute, they have been used in combination to attempt to determine which types of alkylation products are responsible for mutation, transformation, and toxicity. Studies have suggested that the mutation rates produced by these and similar chemicals in cells surviving toxicity correlate well with the number of methyl adducts at the O⁶ position of guanine, but that cytotoxicity (reduced colony-forming efficiency) does not correlate with any single adduct or with the total level of alkylation of DNA. In this study we have investigated the cytotoxic mechanisms of MNNG and MMS in synchronized 10T1/2 cells, using colony-forming ability as a measure of toxicity. Both MNNG and MMS cause dose-dependent reduction in the ability of 10T1/2 cells to produce colonies of more than 50 cells after 2 weeks in culture. MNNG is about 100-fold more toxic than MMS on a molar basis. As indicated by the inability of cells to exclude trypan blue, MMS kills a fraction of the population of treated 10T1/2 cells after a 30-min exposure; the fraction of cells that excludes trypan blue is correlated with dose of MMS and with colony-forming efficiency. Neither the fraction of cells that is permeable to trypan blue nor the relative colony-forming efficiency is affected by the phase of the cycle when 10T1/2 cells are treated with MMS. Furthermore, MMS toxicity for 10T1/2 cells is not potentiated by caffeine, MMS treatment does not delay progress of S phase, and cells that survive acute membrane toxicity complete the cell cycle without significant delay. In contrast, MNNG treatment produces toxicity that is maximal when 10T1/2 cells are exposed during the S phase and the effect of potentiated by caffeine. MNNG treatment delays DNA replication and this delay is reversed by caffeine. In sharp contrast to 10T1/2 cells treated with MMS. MNNG-treated cells are not made permeable to trypan blue, but are blocked in their ability to proliferate. These observations indicate that MNNG and MMS kill 10T1/2 cells by drastically different mechanisms, MNNG producing toxicity mainly by preventing chromosome replication and MMS producing toxicity mainly by damaging cell membranes.     

DNA replication in human cells treated with methyl methanesulfonate

Methyl methanesulfonate (MMS) affects the production of DNA in human cells by reducing the rate of DNA synthesis and by causing the DNA to be synthesized in smaller than normal segments. DNA profiles from alkaline sucrose gradients from cells treated with MMS for 1 h and pulse-labeled at 2.5 h after treatment show more slow-sedimenting DNA than profiles from untreated cells or treated cells pulsed at 0.5 or 4 h after the 1-h treatment. Upon incubation of the pulse-labeled DNA there is an increase in the amount of fast-sedimenting DNA in each sample, indicating repair of the lesions.The amount of DNA synthesized is also reduced 2.5 h after a 1-h treatment but is at near normal levels at 0.5 and 4 h. The reduction in the size of the DNA segments synthesized and the reduction in the rate of DNA synthesis probably reflect the formation and repair of lesions in the parental DNA.     

Independent roles of XRCC1's two BRCT motifs in recovery from methylation damage

XRCC1 is known to be involved in base excision repair (BER)/single-strand break repair (SSBR) through interaction with other BER enzymes. Hypersensitivity of XRCC1-deficient cells against alkylating agents has been explained by loss of interaction with BER proteins. XRCC1 is a unique DNA repair protein containing two BRCT motifs, recently identified in several DNA repair and cell cycle regulating proteins. To study the function(s) of the two BRCT motifs of the XRCC1 protein, we established CHO EM9 (XRCC1-null) cells expressing XRCC1 protein altered in either one of the two BRCT motifs. Colony-forming ability after methyl methanesulfonate (MMS) treatment was dependent on the BRCT-a motif, but not on the BRCT-b motif. Surprisingly, reduced BER/SSBR rate in vivo, measured by an alkaline comet assay, was observed in the BRCT-b motif-deficient cells, while the BRCT-a motif-deficient cells showed the repair rate comparable with the wild-type (WT) cells. The BRCT-a motif-mutated cells, instead, showed deficiency in initiation of DNA replications after MMS treatment. Furthermore, we found that XRCC1 is multiply phosphorylated in vivo and hyperphosphorylation of XRCC1 after MMS treatment is dependent on the BRCT-a motif. These data suggest a new function dependent on the integrity of the BRCT-a motif of XRCC1 in recovery from MMS-induced damage.     

Caffeine-inhibitable control of the radiation-induced G2 arrest in L5178Y-S cells deficient in non-homologous end-joining

The two L5178Y (LY) sublines bear a heterozygous Tp53 mutation that affects its transactivation function. LY-S (radiation-sensitive) cells are deficient in double strand break (DSB) repair by non-homologous end-joining (NHEJ) and do not express p21^WAF1 (Cdkna1) either constitutively or after x-irradiation, in contrast to their radiation-resistant counterpart LY-R cells, which express p21^WAF1 constitutively. Radiation-induced G2 arrest in LY-S cells is very long (11 h/Gy) but 2 mM caffeine treatment shortens it, decreases the fraction of G2 cells and increases the fraction of apoptotic cells. The treatment also increases the DNA damage that is estimated with the comet assay 18 h after irradiation with 5 Gy (ca. 23% of the initial value for x-rays and ca. 47% for x-rays plus caffeine). This indicates that either the repair has not been completed or the apoptotic DNA fragmentation has been initiated (or both). The same treatment applied to x-irradiated (5 Gy) LY-R cells (G2 arrest, 4 h/Gy) has no radiosensitising effect, induces no apoptosis and does not alter the amount of DNA damage left unrepaired (ca. 28%). The results are compatible with the assumption that inhibition of the Atm-dependent homologous recombination repair by caffeine, brings differential effects in LY sublines because of the defect of the alternative DNA repair system (NHEJ) in LY-S cells. Received: 23 June 2000 / Accepted: 5 January 2001     

Methyl methanesulfonate (MMS) produces heat-labile DNA damage but no detectable in vivo DNA double-strand breaks

Homologous recombination (HR) deficient cells are sensitive to methyl methanesulfonate (MMS). HR is usually involved in the repair of DNA double-strand breaks (DSBs) in Saccharomyces cerevisiae implying that MMS somehow induces DSBs in vivo. Indeed there is evidence, based on pulsed-field gel electrophoresis (PFGE), that MMS causes DNA fragmentation. However, the mechanism through which MMS induces DSBs has not been demonstrated. Here, we show that DNA fragmentation following MMS treatment, and detected by PFGE is not the consequence of production of cellular DSBs. Instead, DSBs seen following MMS treatment are produced during sample preparation where heat-labile methylated DNA is converted into DSBs. Furthermore, we show that the repair of MMS-induced heat-labile damage requires the base excision repair protein XRCC1, and is independent of HR in both S.cerevisiae and mammalian cells. We speculate that the reason for recombination-deficient cells being sensitive to MMS is due to the role of HR in repair of MMS-induced stalled replication forks, rather than for repair of cellular DSBs or heat-labile damage.     

Defective flap endonuclease 1 activity in mammalian cells is associated with impaired DNA repair and prolonged S phase delay.

Flap endonuclease 1 (FEN-1) is a 5'-3' flap exo-/endonuclease that plays an important role in Okazaki fragment maturation, nonhomologous end joining of double-stranded DNA breaks, and long patch base excision repair. Here, we demonstrate that the wild type FEN-1 binds tightly to chromatin in conjunction with proliferating cell nuclear antigen (PCNA) recruitment after MMS treatment, and the nuclease-defective FEN-1 increased the sensitivity of the cells to methylmethane sulfonate (MMS) and to UV light but not to ionizing radiation. In contrast, the cells expressing the nuclease-defective and PCNA binding-defective double mutant FEN-1 exhibited sensitivities similar to those in the cells expressing the wild type FEN-1. MMS treatment caused a prolonged delay of S phase progression and impairment in colony-forming activity of cells expressing nuclease-defective FEN-1. A comet assay demonstrated that DNA repair after MMS or UV treatment was impaired in the cells expressing nuclease-deficient FEN-1 but not in the cells with double-mutated FEN-1. Taken together, these findings suggest that FEN-1 plays an essential role in the DNA repair processes in mammalian cells and that this activity of FEN-1 is PCNA-dependent.     

Physiological Modifications in the Production and Repair of Methyl Methane Sulfonate-Induced Breaks in the Deoxyribonucleic Acid of Escherichia coli K-12

The medium in which Rec(+) strains of Escherichia coli K-12 are grown affected their sensitivity to treatment with methyl methane sulfonate (MMS). Rec(+) cells grown to the stationary phase in glucose-enriched nutrient broth (GNB) were more resistant to MMS than cells grown in nutrient broth (NB). The repair of MMS-induced breaks (or alkali-labile bonds) in the deoxyribonucleic acid (DNA) from E. coli K-12 strains AB1157, AB1886 uvrA6, and SR111 recA13 recB21 grown in GNB and NB media was examined by means of alkaline sucrose gradient centrifugation. It appeared that essentially all of the repair of breaks that occurred, as evidenced by an increase in "molecular weight," took place within 10 min after treatment with MMS under our conditions. Cell survival was highest in cells for which the size of the DNA after the post-treatment incubation was the largest. The largest DNA after post-treatment incubation was found in Rec(+) cells grown in GNB medium. The results suggest that these cells may have an enhanced capacity for repairing breaks in DNA.     

DNA polymerase III requirement for repair of DNA damage caused by methyl methanesulfonate and hydrogen peroxide.

The pcbA1 mutation allows DNA replication dependent on DNA polymerase I at the restrictive temperature in polC(Ts) strains. Cells which carry pcbA1, a functional DNA polymerase I, and a temperature-sensitive DNA polymerase III gene were used to study the role of DNA polymerase III in DNA repair. At the restrictive temperature for DNA polymerase III, these strains were more sensitive to the alkylating agent methyl methanesulfonate (MMS) and hydrogen peroxide than normal cells. The same strains showed no increase in sensitivity to bleomycin, UV light, or psoralen at the restrictive temperature. The sensitivity of these strains to MMS and hydrogen peroxide was not due to the pcbAl allele, and normal sensitivity was restored by the introduction of a chromosomal or cloned DNA polymerase III gene, verifying that the sensitivity was due to loss of DNA polymerase III alpha-subunit activity. A functional DNA polymerase III is required for the reformation of high-molecular-weight DNA after treatment of cells with MMS or hydrogen peroxide, as demonstrated by alkaline sucrose sedimentation results. Thus, it appears that a functional DNA polymerase III is required for the optimal repair of DNA damage by MMS or hydrogen peroxide.     

Repair of 3-methyladenine and 7-methylguanine in nuclear DNA of Chlamydomonas: Requirement for protein synthesis

The removal of 3-methyladenine and 7-methylguanine from nuclear DNA was determined following exposure of Chlamydomonas reinhardi to methyl methanesulfonate (MMS). The amount of 3-methyladenine in DNA was determined using an extract from Micrococcus luteus that has a 3-methyladenine-DNA glycosylase. The amount of 7-methylguanine was estimated by heating the DNA for 30 min at 70° followed by alkaline hydrolysis of the resulting apurinic sites. The molecular weight of the DNA was determined using alkaline sucrose gradients. The 3-methyladenine is removed with a half-life of 2–3 h whereas the 7-methylaguanine is removed with a half-life of 10–12 h. The rate of removal of the 7-methylguanine is more than an order of magnitude faster than the estimated non-enzymatic hydrolysis rate indicating the probability of enzymatic repair. Addition of cycloheximide immediately after MMS treatment inhibits the removal of 3-methyladenine and 7-methylguanine from DNA. If cycloheximide is added 1.5 h after treatment with MMS, there is much less inhibition of the removal of 3-methyladenine. These results are interpreted to mean that MMS induces the synthesis of 1 or more proteins that are required for the repair of 3-methyladenine from Chlamydomonas DNA.     

Caffeine-induced enhancement of chromosome damage in human lymphocytes treated with methyl-methanesulphonate, mitomycin C and X-rays

Two hypothese have been put forward in the literature to explain the synergistic effect of caffeine with several mutagens: (1) binding of caffeine to DNA, and (2) inhibition of DNA repair.Autoradiographic studies with ³H- and ¹⁴C-labelled caffeine did not support the binding hypothesis. Caffeine enchanced in a synergistic way the amount of chromatid breaks and exchanged induced in human lymphocytes with methyl-methanesulphonate (MMS), mitomycin C (MC) and X-rays. The results are best explained if caffiene inhibits a post-replication repair process, particularly the filling-in of gaps in the newly synthesized DNA.     

Studies on DNA repair in early spermatid stages of male mice after in vivo treatment with methyl-, ethyl-, propyl-, and isopropyl methanesulfonate.

In vivo DNA repair occurring in early spermatid stages of the mouse has been studied with four mutagens that are chemical homologs: MMS, EMS, PMS and IMS. Using the well-studied sequence of events that occurs during spermatogenesis and spermiogenesis in the mouse, aatids was measured by the unscheduled incorporation of [3H]dT into these germ cells which were recovered from the caudal epididymides 16 days after chemical treatment. Purification of the caudal sperm DNA at this time verified that the [3H]dT was incorporated into the DNA. For each chemical mutagen a study was made on the level of DNA repair occurring in early spermatids as a function of the administered, in vivo dose. Within experimental errors, all four chemicals produced a linear increase in DNA repair in early spermatids with increasing dose. Only the highest dose of MMS (100 mg/kg) produced a greater repair response than expected for a linear curve. At equimolar doses the most effective chemical in inducing DNA repair was MMS, followed by EMS, IMS and PMS. When testicular injections of [3H]dT were given at the same time as the intraperitoneal injections of the mutagens, the amount of unscheduled incorporation of [3H]dT into the DNA of early spermatids was maximized. Since [3H]dT has been shown to be available for incorporation into germ-cell DNA for only approximately 1 h after injection, all four mutagens must reach the DNA of early spermatids and begin producing "repairable" lesions within 1 h after treatment. The amount of DNA repair occurring at later times after chemical treatment of early spermatids was studied by testicular injections of [3H]dT 1/2, 1, 2 and 3 days after chemical treatment. Repair was still occurring in the early spermatids at 3 days post-treatment; this repair is most likely a manifestation of the finite rate of the repair process rather than resulting from newly alkylated DNA. For MMS and EMS there was a rapid decrease in the level of DNA repair in the first 1/2 day following treatment. This was followed by a much slower, exponential decrease in the level of repair out to 3 days post-treatment. The curves suggest that the amount of repair is proportional to the number of repairable lesions still present in the DNA. For PMS and IMS the level of repair decreases rapidly in the first 1/2 day after treatment and thereafter remains relatively constant through 3 days post-treatment. With all four mutagens, DNA repair in early spermatids was detectable at doses 5 to 10 times lower than those required to observe other genetic end points such as dominant lethals, translocations and specific-locus mutations in any germ-cell stage. The sensitivity of detection of in vivo DNA repair in the germ cells of male mice makes such a system a useful adjunct to other genetic tests for studying chemical mutagenesis in mammals.     

Increased integration of viral genome following chemical and viral treatment of hamster embryo cells.

Treatment of hamster embryo cells with diverse classes of chemical carcinogens enhances transformation by a carcinogenic simian adenovirus, SA7. Virus transformed foci selected from plates pretreated with 3-methyl-cholanthrene (MCA), methyl methanesulfonate (MMS) or 7,12-dimethylbenz[a]anthracene (DMBA) and established as cell lines in culture, contained equivalent amounts of SA7 viral genome. However, hamster embryo cultures treated with MMS or nickel sulfate had increased amounts of SA7 DNA integrated into cellular DNA when examined 2--9 days after chemical treatment and viral inoculation. An increased uptake of SA7 DNA was demonstrated in hamster cells treated with MMS during DNA repair synthesis in cells retricted in scheduled DNA synthesis by amino acid deprivation; addition of virus after the repair period did not result in an increased integration of viral DNA. These data suggest that enhancement of viral oncogenesis by chemical carcinogens or mutagens may be related to the formation of additional attachment sites in cellular DNA for insertion of viral DNA, thereby increasing the probability of viral transformation.