Mutation Research, 275 (1992) 1-6 © 1992 Elsevier Science Publishers B.V. All rights reserved 0921-8734/92/$05.00
!
MUTAG! 09075
The ability of liver extracts from different-aged rats to repair 'mis-instructive' and 'non-instructive' lesions of DNA C. P a r d i n i , L. M a r i a n i , M. Voliani, G. R a i n a l d i a n d L. Citti lstituto di Mutagenesi e Differenziamento CNR, Pisa (Italy) (Received 16 September 1990) (Accepted 14 August 1991)
Keywords: O6-Methylguanine; DNA damage: Apurinic/apyrimidinic sites; (Rats)
Summary The ability to repair 'mis-instructive', O6-methylguanine, and 'non-instructive', AP sites, DNA lesions in Fischer 344 rat livers at various ages was determined. Different behaviours were observed. While the AP-endodesoxyribonuclease enzymes displayed a high constant level throughout the animals' lifetime, the O6-methylguanine-DNA methyltransferase activity presented a stepwise modulation (DNA normaiisation of results): the O6-MT activity significantly increased within the first month of animal life and enhanced again after 6 months reaching a maximum plateau in the 12-18-month-old animals. Thereafter a net significant decrease of O~-MT enzyme was detected in the 24-month-old group. While the repair of the widely formed AP sites appeared uniformly efficient like 'house keeping' functions, the removal of the rare precancerous O6-methylguaninc is age.dependent indicating a decreased protection of the youngest and oldest animals against this 'mis.instructive' damage. However, any extrapolation of the age-associated cancer risk needs further assessment.
The cumulative cancer risk in mammals increases with age and is inversely correlated with species life span. This relation is based on the assumption that damages induced by metabolic transformations accumulate in DNA contributing to both the involutive process of tissue senescence and the cancer development (Kirkwood, 1989; Franceschi, 1989; Newell et al., 1989). Among the various damaging agents, endogenous stressors such as aikylating agents and oxygen radicals have been indicated to produce DNA
Correspondence: Dr. L. Cirri, Istituto di Mutagenesi e Differenziamento CNR, via Svezia 10/2a, 1-56124 Pisa (Italy).
lesions responsible for different effects (Oensler and Bernstein, 1981; Ames, 1989a). The biological systems have developed a variety of defence mechanisms which enable cells to cope with endogenous or exogenous damaging agents. In this respect, DNA repair plays a crucial role in removing the harmful lesions that have escaped previous defence functions (Hart and Setlow, 1974; Vijg and Knook, 1987). Thus a possible decrease in DNA-repair ability during age could increase the initiating potency of carcinogenic agents. Many studies have been done in order to correlate DNA repair and longevity of various species, but the resulting interspecies correlations appear conaplex depending on the par-
ticular tissue as well as on the considered repair process (Kirkwood, 1989). Moreover, studies on age-related repair differences have been performed considering bulky DNA lesions, mainly pyrimidine dimers (Hart and Setlow, 1974; Vijg and Knook, 1987; Mullaart et al., 1989). Less is known about the relations between ageing and the repair of lesions that produce little, if any, distortion of the DNA template structure. Since such DNA modifications are frequently induced by various stressors and accumulate in DNA (Ames, 1989b; Park and Ames, 1988; Randerath et al., 1986), the relations of these with senescence processes should be investigated. This report deals with the age dependence of 2 DNA-repair activities in the rat liver. These activities are representative of 2 relevant classes of lesions involved in different biological responses: O~-methylguanine (O~-m-gua), a typical 'mis-instructive' lesion, and apurinic/apyrimidinic sites (AP sites), which represent 'non-instructive' lesions. While the former induces mainly point mutations after the replicative synthesis of DNA, and is recognised as a critical precancerous lesion, the latter causes a block of DNA replication and induces many post-replicative rearrangements (Eadie et al., 1984; Saffllili et al., 1985; Schaaper et al., 1982; Kaufmann, 1989). O~'-m-gua is repaired by Ot'-methylguanine-DNA mcthyltransferase (Or'-MT), an enzyme that in a suicide stoichiometric fashion removes the methyl group from the guanine moiety restoring an intact DNA strand. AP sites are cleaved by specific repair enzymes, the endodesoxyribonucleases (APendo), producing single-strand breaks which in turn are the substrates for the next steps of the excision repair pathway. Here we show that, in rat liver, the Ot'-MT activity displays an age-dependent modulation while the AP-endo are uniformly active. Materials and methods
Animals Male Fischer 344 inbred rats (Charles River, Italy) were maintained under standard housing conditions with free access to food and water until they were killed. Livers, obtained after rapid dissection, were frozen in liquid nitrogen and
then stored at - 80°C. In each set of experiments, fresh liver specimens were employed for extract preparation.
Extract preparation Whole liver homogenates were prepared as described by Gerson et al. (1986), using an Ultra-turrax homogenizer. After sonication of the mixture, samples were centrifuged at 10,000 × g for 2 min to remove cellular debris and the recovered supernatants were frozen and stored at -80°C until use. Alternative crude nuclear extracts were obtained after sucrose sedimentation of liver tissue nuclei as already described (Citti et al., 1990). Extracts were characterised for DNA and protein concentrations by bisbenzimide H 33258 fluorochrome DNA binding and Bio-Rad protein assay respectively.
06-Methylguanine.DNA methyltransferase assay The O6-MT assay was performed, as previously reported (Citti et al., 1987), using a methylated DNA substrate containing O~-[aH]methylguanine prepared according to the method described by Wiestler et al. (1984). An alternative simplified technique, developed by Myrnes ¢t al. (1984), was also employed especially to assay the whole crude homogenates. Briefly, the reaction mixture was treated with 2.5 vol of cold 6% TCA; the DNA substrate, included in the precipitate, was then hydrolysed by heating at 80°C in order to solubilise the interfering radioactivity. After BSA carrier addition, the proteic precipitate was collected on glass fibre filter (Whatman GF/C). Filters, washed and dried, were treated with a tissue solubiliser (Soluene-350, Packard) and counted for radioactivity using a scintillation cocktail (Hionic-Fluor, Packard).
Apurinic / apyrimidinic endodesoxyribonuclease as. say A synthetic partially apurinic poly(dA-dT) substrate ['~H]methyl-labelled on thymidine was employed (poly(dA-dT)-AP). The depurinated substrate was prepared according to the method developed by Talpaert-Borl6 et al. (1983). Briefly, the polymer was reacted 1 h at 3"PC with 0.5 M methyl methanesulphonate in a buffered solution
at pH 7. At the end the alkylated poly(dA-dT) was precipitated by cold ethanol, dialysed in standard saline citrate solution (0.1 x ), and then heat-depurinated at 40°C for 18 h. This treatment produced an average of 50-60 AP sites/1000 nucleotides, determined by acid-soluble fraction as reported below. The overall DNA fragmentation at AP sites by the AP-endo enzymes was determined to be the acid-soluble fraction after perchloric acid precipitation of poly(dA-dT)-AP applying the curve developed by Bricteu,x-Gr6goire et al. (1986) which correlates the soluble fraction mid the number of single-strand breaks. 2-/~g aliquots of the poly(dA-dT)-AP substrate, containing approximately 700 pmole of AP sites, were reacted in a 50-/,d incubation mixture containing 2 mM MgCl 2, 0.15 M NaCl, 0.015 M sodium citrate, and nuclear extracts (10-20 mg proteins). Samples were incubated for different times. At the end, the DNA carrier, saturated NaCI, and perchloric acid were added in order to precipitate the apurinic DNA. The supernatants were collected after 10,000 x g centrifugation and assayed by liquid scintillation counting for soluble radioactivity determination. Blanks were obtained by incubation of identical samples without extracts. References were performed by reacting extract-incubated samples with 0.4 N NaOH at 37°C for 30 min in order to assess any possible degradation of substrate over the theoretical fragmentation after the complete alkaline cleavage of AP sites. A typical time course of enzymatic incubation is represented in Fig. 1. In the figure are also reported the curves corresponding to the blanks and reference samples representing the spontaneous and the overall (also aspecific) substrate fragmentation respectively. Results
The effect of age on the hepatic O~-MT activity in Fischer 344 rats was movitored using both nuclear and whole crude preparations (data not shown). No differences were obtained when extracts were prepared from fresh or frozen specimens. Fig. 2A shows the results obtained using nuclear and total liver homogenates. The activities, calculated on the basis of extract DNA
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Fig. 1. Cleavage kinetics of the AP sites. The cleavage of poly(dA-dT)-AP substrate measured as acid-soluble radioactive fraction. Kinetics of apurinic polymer fragmentation by extract incubations ( I ) . Reference samples treated with NaOH after extract incubations (overall aspecific cleavage) ( • ) . Spontaneous polymer fragmentation (blanks)(0).
content, showed similar-shaped relations between repair and the age of the animals. We observed a stepwise modulation of transferase levels
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Fig. 2. Age dependence of the O°-methylguanine-DNAmethyltransferase activity in rat liver extracts. Whole crude liver homog.enates (o): nuclear crude liver homogenates (*). (A) Data calculated on the basis of extract DNA concentration. (B) Data calculated on the basis of extract protein concentration. Each point represents the mean of 10-15 independent measures obtained from at least 6 animals per group. Vertical lines represent the standard deviation.
throughout the animals' lifetime: values ranged from 1.51 pmole/mg DNA in the youngest (1 week) to 7.44 p m o l e / m g DNA in 18-month-old rats using nuclear homogenates, or from 3.8 p m o l e / m g DNA to 12.98 pmole/mg DNA using crude whole extracts. The observed differences of O6-MT activity between 1-week- and 1-month-old animal groups as well as between 6- and 12- or 18- and 24-month-old groups were highly significant (P < 0.01) as measured by Student's t-test (see figure legend). Transferase values, constantly higher using whole homogenates rather than nuclear preparations, reflect a contribution of the cytosolic fraction to the overall O6-MT activity. When transferase activities were calculated on the basis of extract protein concentration (Fig. 2B), a considerable variability was obtained. Consequently no significant relationship between age and O6-MT activity was detected. The cleavage of AP sites by specific endodesoxyribonucleases (AP-endo) was determined using only nuclear crude homogenates because the degradative cytosolic enzymes quickly destroyed the synthetic apurinic substrate (data not shown). The data obtained by nuclear hepatic preparations calculated with respect to DNA or protein extract concentrations are reported in Fig. 3. No significant differences in the repair of AP sites DO,
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AGE (months)
Fig. 3. Age dependence of the AP-endodeoxyribonudease
activityin crude nuclearrat liverextracts. (A) Data calculated on the basis or extract DNA concentration,(B) Data calculated on the basisof extract protein concentration.Each point represents the mean of 8-10 independentmeasuresobtained from at least 6 animals per group, Vertical lines represents the standard deviation,
was detected. The activity measures ranged close to the mean values: 11.45 nmole/(min × mg DNA) or 1.90 nmole/(min × mg protein). Discussion
The repair of AP sites and O6-m-gua in the liver of Fischer 344 rats displays quite different behaviours throughout the lifetime of the animals. So the mechanisms involved in the control of these activities appear independently regulated in the ageing. It was observed that AP-endo activities are invariably present at high constitutive levels during the lifetime of the animals. Because the AP sites represent a DNA lesion that is widely produced spontaneously and by a considerable number of agents, it is not surprising that the AP-endo activities are expressed like 'housekeeping' gene products in order to assure normal tissue development. By contrast, the O~-MT activity appears to be age-related only when the results are expressed as a function of DNA concentration of extracts. This normalisation of measures seemed more appropriate to us than protein normalisation because the number of cells per gram of tissue can vary greatly among different animals and among the different stages of tissue development. Consequently, higher data variability obtained using protein parameters, made it impossible for us to measure any significant difference in the O~.MT levels, like Woodhead et al. (1985) or Nakatsuru et al, (1989). The O~-MT enhancement observed within the first month of animal life, using DNA normalisation, could be linked to the liver maturity reached with the normal diet after weaning. We also detected an increase in enzyme until the maximal plateau levels, but only a net decrease of repair activity for the oldest rats was observed, indicating a reduced ability of aged animals to counteract a 'mis-instructive' lesion eventually produced on DNA. The modulation of O6.AT activity accords with findings described in rats by Likhachev et al. (1985) and with similar observations obtained in mice by Nakatsuru et al. (1989). From these results it appears that the susceptibility of rat liver to alk'ylating agents could be higher in the youngest and in the oldest animals. Nevertheless, extrapolations of the age-associated spon-
taneous tumour incidence need further assessment. Moreover, the inducibility of O6-AT by a number of different agents may suggest an important role of this repair activity in ageing. In fact O6-AT has been reported to be induced, in aerobic conditions, by X rays, y rays or bleomycin, indicating a possible involvement of "OH radicals (Margison et al., 1985; Sehmerold and Spath, 1986). This enzyme modulation could represent a nuclear marker for the cumulative damage by active oxygen, and O6-AT could be important in the cell defence perhaps as radical scavenger (yon Hofe and Kennedy, 1988). On the other hand the age-related accumulation of aikyl damages in the DNA o," animals (Ames, 1989) could be crucial in old rats. The less effective O6-m-gua repair could contribute, besides the induction of harmful mutations, to the modification of 5-methyicytosine patterns, as observed in aging (Ono et al., 1989; Drinkwater et al., 1989), producing possible alterations of gene expression. Acknowledgement This work was partially supported by the Italian Association for Cancer Research (AIRC).
References Ames, B.N, (198%) Mulagenesis and carcinogenesis: endogenous and exogenous factors, Environ, Mol. Mutagen., 14, 66-77. Ames, B.N. (1989b) Endogenous DNA damage as related to cancer and aging, Mutation Res., 214, 41-46. Bricteux-Gr~goire, S,, M. Liuzzi, M. Talpaert.Borl~, M. Winand and W.G. Verly (1986) Relationship between DNA acid-solubility and frequency of single-strand breaks near apurinic sites, Biochim. Biophys. Acta, 867, 24-30. Citti, L., L. Mariani, R. Fiorio and G. Malvaldi (1987) Increased DNA repair ability of the rat hepatocyte nodules, Med. Sci. Res., 15, 429-430. Citti L., L. Mariani, M. Mengozzi and G. Malvaldi (1990) DNA repair systems in early and persistent hepatoeyte nodules in the rat, J. Cancer Res. Clin. Oncol., 116, 156-158. Drinkwater, R.D., T.J. Blake, A.A. Morley and D.R. Turner (1989) Human lymphocytes aged in vivo have reduced levels of methylation in trascriptionally active and inactive DNA, Mutation Res., 219, 29-37.
Eadie, J.S., M. Conrad, D. Toorchen and M.D. Topoi iag/~4D Mechanism of mutagenesis by O%metbylguanine, Nature, 308, 201-203. Franceschi, C. (1989) Cell proliferation, cell death and aging, Aging, 1, 1-14. Gensler, H.L., and H. Bernstein (198!) DNA damage as the primary cause of ageing, Q. Rev~ Bf,oL, 56, 279-3113.. Hart, R.W., and R.B. Setlow (1974j Correlation bel~een deoxyribonucleic acid excision repair and lifespan in a number of mammalian species, Prec. Natl. Acad. Sci. (U.S.A.), 71, 2!69-2173. Kaufmann, W.K. (1989) Pathways of h~man cell posl-repl~cation repair, Carcinogenesis, 10, t-11. Kirk'wood, T.B.L. (1989) DNA, mutations and aging, Mulalion Res., 219, 1-7. Likhachev, A.J. (1985) Effect of age on DNA-repair in carcinogenesis due to alkylaling agents, in: A. Likhachev~ V. Anisimov and R. Montesano (Ed~s.), Age-Related Factors in Carcinogenesis, IARC Sci. Publ. No. 58, lnternJationaU Agency for Research on Cancer, Lyon, pp. 239-25t. Margison, G.P., J. Butler and B. Hoey (1985) O*-methylguanine methyltransferase actiuity is increased in rat tissues by ionising radiations, Carcinogenesis, 6, 1699-1702. Mullaart, E., M.E.T.I. Boerrigter, P.H.M. Lehman and JL Viilg (1989) Age-related induction and disappearance of carcinogen-DNA-adducts in livers of rats exposed ~:o le~ levels of 2-acetylaminofluorene, Chem.-Biol. Interact., 69, 373-384. Myrne,% B., K. Norstrand, K.E. Giercksky, C. Sjunneskog and H. Krokan (1984) A simplified assay for O~-meth~fb guanine-DNA methyltransferase activity and its a:l~plic~lion to human neoplastic and non-neoplastic 1issues, Carcinogenesis, 5, 1061-1064. Nakatsura, Y., K. Aoki and T. Ishik~twa (1989) Age and s~rain dependence of O~-methylguan:ine DNA methyllransf, erase activity in mice, Mutation Res., 2t9, 51-56. Newell, G,R,, M.R. Spitz and J,G, Sider (1989} Canc.¢r and age, Semin. Oncol., 16, 3-9. One, T., N, Takahashi and S. O]~,da 11989) Age-assec~ialerdl changes in DNA methylation and mRNA level e,[ the c.myc gene in spleen and liver of mice, Mutatien Res, 219, 39-50. Park, J.W., and B.N. Ames (1988)7-methyiguanine addt~cts i~ DNA are normally present at high levels and increase o~ aging: analysis by HPLC with electrochemical det:ection, Prec. Natl. Acad. Sci. (U.S.A.), 85, 7467-7470. Randerath, K., M.V. Reddy and R.M. Disher (1986) Age- .~n~ tissue-related DNA modifications in untreated rats: detection by .~2P-postlabeling assay and possible significance for spontaneous tumor induction and aging, Carcinogenesis, 7, 1615-1617. Saffhill, R., G.P. Margison and PJ. O'Connor (I985)Mechanisms of carcinogenesis induced by alkylating agents, Biochim. Biophys. Acta, 823, 1 [ I - 145. Schaaper, R.M., B.W. Glickman and L.A. Loeb (19S2~ Role of depurination in mutagenesis by chemical carcinogens~ Cancer Res., 42, 3480-3485.
Schmerold, !., and A. Spath (1986) Induction of rat liver O6-alkylguanine-DNA alkyltransferase by bleomycin, Chem.-Biol. Interact., 60, 297-304. Talpaert-Borl6, M., and M. Liuzzi {1983) Reaction of apurinic/apyrimidinic sites with [14C]methoxyamilie: a method for the quantitative assay of AP sites in DIqA, Biochim. Biophys. Acta, 740, 410-416. Vijg, J., and D.L. Knook (1987) DNA-repair in relation to the aging process, J. Am. Geriatr. Soc., 35, 532-541. yon Hofe, E., and A.R. Kennedy (1988) In vivo induction of
O6-methylguanine-DNA methyltransferase in C3H/ 10TI/2 cells by X-rays is inhibited by nitrogen, Carcinogenesis, 9. 679-681. Wiestler, O., P. Kleihues and A.E. Pegg (1984) O6-alkylguanine-DNA alkyltfansferase activity in human brain and brain tumors, Carcinogenesis, 5, 121-124. Woodhead, A.D., B.J. Merry, E.H. Cao, A.M. Holehan, E. Grist and C. Carlson (1985) Levels of O6-methylguanine acceptor protein of rats and their relationship to carcinogenicity and aging, J. Natl. Cancer Inst., 75, 1141-1145.
!
MUTAG! 09075
The ability of liver extracts from different-aged rats to repair 'mis-instructive' and 'non-instructive' lesions of DNA C. P a r d i n i , L. M a r i a n i , M. Voliani, G. R a i n a l d i a n d L. Citti lstituto di Mutagenesi e Differenziamento CNR, Pisa (Italy) (Received 16 September 1990) (Accepted 14 August 1991)
Keywords: O6-Methylguanine; DNA damage: Apurinic/apyrimidinic sites; (Rats)
Summary The ability to repair 'mis-instructive', O6-methylguanine, and 'non-instructive', AP sites, DNA lesions in Fischer 344 rat livers at various ages was determined. Different behaviours were observed. While the AP-endodesoxyribonuclease enzymes displayed a high constant level throughout the animals' lifetime, the O6-methylguanine-DNA methyltransferase activity presented a stepwise modulation (DNA normaiisation of results): the O6-MT activity significantly increased within the first month of animal life and enhanced again after 6 months reaching a maximum plateau in the 12-18-month-old animals. Thereafter a net significant decrease of O~-MT enzyme was detected in the 24-month-old group. While the repair of the widely formed AP sites appeared uniformly efficient like 'house keeping' functions, the removal of the rare precancerous O6-methylguaninc is age.dependent indicating a decreased protection of the youngest and oldest animals against this 'mis.instructive' damage. However, any extrapolation of the age-associated cancer risk needs further assessment.
The cumulative cancer risk in mammals increases with age and is inversely correlated with species life span. This relation is based on the assumption that damages induced by metabolic transformations accumulate in DNA contributing to both the involutive process of tissue senescence and the cancer development (Kirkwood, 1989; Franceschi, 1989; Newell et al., 1989). Among the various damaging agents, endogenous stressors such as aikylating agents and oxygen radicals have been indicated to produce DNA
Correspondence: Dr. L. Cirri, Istituto di Mutagenesi e Differenziamento CNR, via Svezia 10/2a, 1-56124 Pisa (Italy).
lesions responsible for different effects (Oensler and Bernstein, 1981; Ames, 1989a). The biological systems have developed a variety of defence mechanisms which enable cells to cope with endogenous or exogenous damaging agents. In this respect, DNA repair plays a crucial role in removing the harmful lesions that have escaped previous defence functions (Hart and Setlow, 1974; Vijg and Knook, 1987). Thus a possible decrease in DNA-repair ability during age could increase the initiating potency of carcinogenic agents. Many studies have been done in order to correlate DNA repair and longevity of various species, but the resulting interspecies correlations appear conaplex depending on the par-
ticular tissue as well as on the considered repair process (Kirkwood, 1989). Moreover, studies on age-related repair differences have been performed considering bulky DNA lesions, mainly pyrimidine dimers (Hart and Setlow, 1974; Vijg and Knook, 1987; Mullaart et al., 1989). Less is known about the relations between ageing and the repair of lesions that produce little, if any, distortion of the DNA template structure. Since such DNA modifications are frequently induced by various stressors and accumulate in DNA (Ames, 1989b; Park and Ames, 1988; Randerath et al., 1986), the relations of these with senescence processes should be investigated. This report deals with the age dependence of 2 DNA-repair activities in the rat liver. These activities are representative of 2 relevant classes of lesions involved in different biological responses: O~-methylguanine (O~-m-gua), a typical 'mis-instructive' lesion, and apurinic/apyrimidinic sites (AP sites), which represent 'non-instructive' lesions. While the former induces mainly point mutations after the replicative synthesis of DNA, and is recognised as a critical precancerous lesion, the latter causes a block of DNA replication and induces many post-replicative rearrangements (Eadie et al., 1984; Saffllili et al., 1985; Schaaper et al., 1982; Kaufmann, 1989). O~'-m-gua is repaired by Ot'-methylguanine-DNA mcthyltransferase (Or'-MT), an enzyme that in a suicide stoichiometric fashion removes the methyl group from the guanine moiety restoring an intact DNA strand. AP sites are cleaved by specific repair enzymes, the endodesoxyribonucleases (APendo), producing single-strand breaks which in turn are the substrates for the next steps of the excision repair pathway. Here we show that, in rat liver, the Ot'-MT activity displays an age-dependent modulation while the AP-endo are uniformly active. Materials and methods
Animals Male Fischer 344 inbred rats (Charles River, Italy) were maintained under standard housing conditions with free access to food and water until they were killed. Livers, obtained after rapid dissection, were frozen in liquid nitrogen and
then stored at - 80°C. In each set of experiments, fresh liver specimens were employed for extract preparation.
Extract preparation Whole liver homogenates were prepared as described by Gerson et al. (1986), using an Ultra-turrax homogenizer. After sonication of the mixture, samples were centrifuged at 10,000 × g for 2 min to remove cellular debris and the recovered supernatants were frozen and stored at -80°C until use. Alternative crude nuclear extracts were obtained after sucrose sedimentation of liver tissue nuclei as already described (Citti et al., 1990). Extracts were characterised for DNA and protein concentrations by bisbenzimide H 33258 fluorochrome DNA binding and Bio-Rad protein assay respectively.
06-Methylguanine.DNA methyltransferase assay The O6-MT assay was performed, as previously reported (Citti et al., 1987), using a methylated DNA substrate containing O~-[aH]methylguanine prepared according to the method described by Wiestler et al. (1984). An alternative simplified technique, developed by Myrnes ¢t al. (1984), was also employed especially to assay the whole crude homogenates. Briefly, the reaction mixture was treated with 2.5 vol of cold 6% TCA; the DNA substrate, included in the precipitate, was then hydrolysed by heating at 80°C in order to solubilise the interfering radioactivity. After BSA carrier addition, the proteic precipitate was collected on glass fibre filter (Whatman GF/C). Filters, washed and dried, were treated with a tissue solubiliser (Soluene-350, Packard) and counted for radioactivity using a scintillation cocktail (Hionic-Fluor, Packard).
Apurinic / apyrimidinic endodesoxyribonuclease as. say A synthetic partially apurinic poly(dA-dT) substrate ['~H]methyl-labelled on thymidine was employed (poly(dA-dT)-AP). The depurinated substrate was prepared according to the method developed by Talpaert-Borl6 et al. (1983). Briefly, the polymer was reacted 1 h at 3"PC with 0.5 M methyl methanesulphonate in a buffered solution
at pH 7. At the end the alkylated poly(dA-dT) was precipitated by cold ethanol, dialysed in standard saline citrate solution (0.1 x ), and then heat-depurinated at 40°C for 18 h. This treatment produced an average of 50-60 AP sites/1000 nucleotides, determined by acid-soluble fraction as reported below. The overall DNA fragmentation at AP sites by the AP-endo enzymes was determined to be the acid-soluble fraction after perchloric acid precipitation of poly(dA-dT)-AP applying the curve developed by Bricteu,x-Gr6goire et al. (1986) which correlates the soluble fraction mid the number of single-strand breaks. 2-/~g aliquots of the poly(dA-dT)-AP substrate, containing approximately 700 pmole of AP sites, were reacted in a 50-/,d incubation mixture containing 2 mM MgCl 2, 0.15 M NaCl, 0.015 M sodium citrate, and nuclear extracts (10-20 mg proteins). Samples were incubated for different times. At the end, the DNA carrier, saturated NaCI, and perchloric acid were added in order to precipitate the apurinic DNA. The supernatants were collected after 10,000 x g centrifugation and assayed by liquid scintillation counting for soluble radioactivity determination. Blanks were obtained by incubation of identical samples without extracts. References were performed by reacting extract-incubated samples with 0.4 N NaOH at 37°C for 30 min in order to assess any possible degradation of substrate over the theoretical fragmentation after the complete alkaline cleavage of AP sites. A typical time course of enzymatic incubation is represented in Fig. 1. In the figure are also reported the curves corresponding to the blanks and reference samples representing the spontaneous and the overall (also aspecific) substrate fragmentation respectively. Results
The effect of age on the hepatic O~-MT activity in Fischer 344 rats was movitored using both nuclear and whole crude preparations (data not shown). No differences were obtained when extracts were prepared from fresh or frozen specimens. Fig. 2A shows the results obtained using nuclear and total liver homogenates. The activities, calculated on the basis of extract DNA
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FJ. . . . . . .
11.
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0
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6
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Fig. 1. Cleavage kinetics of the AP sites. The cleavage of poly(dA-dT)-AP substrate measured as acid-soluble radioactive fraction. Kinetics of apurinic polymer fragmentation by extract incubations ( I ) . Reference samples treated with NaOH after extract incubations (overall aspecific cleavage) ( • ) . Spontaneous polymer fragmentation (blanks)(0).
content, showed similar-shaped relations between repair and the age of the animals. We observed a stepwise modulation of transferase levels
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"? 24
Fig. 2. Age dependence of the O°-methylguanine-DNAmethyltransferase activity in rat liver extracts. Whole crude liver homog.enates (o): nuclear crude liver homogenates (*). (A) Data calculated on the basis of extract DNA concentration. (B) Data calculated on the basis of extract protein concentration. Each point represents the mean of 10-15 independent measures obtained from at least 6 animals per group. Vertical lines represent the standard deviation.
throughout the animals' lifetime: values ranged from 1.51 pmole/mg DNA in the youngest (1 week) to 7.44 p m o l e / m g DNA in 18-month-old rats using nuclear homogenates, or from 3.8 p m o l e / m g DNA to 12.98 pmole/mg DNA using crude whole extracts. The observed differences of O6-MT activity between 1-week- and 1-month-old animal groups as well as between 6- and 12- or 18- and 24-month-old groups were highly significant (P < 0.01) as measured by Student's t-test (see figure legend). Transferase values, constantly higher using whole homogenates rather than nuclear preparations, reflect a contribution of the cytosolic fraction to the overall O6-MT activity. When transferase activities were calculated on the basis of extract protein concentration (Fig. 2B), a considerable variability was obtained. Consequently no significant relationship between age and O6-MT activity was detected. The cleavage of AP sites by specific endodesoxyribonucleases (AP-endo) was determined using only nuclear crude homogenates because the degradative cytosolic enzymes quickly destroyed the synthetic apurinic substrate (data not shown). The data obtained by nuclear hepatic preparations calculated with respect to DNA or protein extract concentrations are reported in Fig. 3. No significant differences in the repair of AP sites DO,
A
t),
J
/E
Q
0 j ,......................
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o
4
e
))
16
io
~a
AGE (months)
Fig. 3. Age dependence of the AP-endodeoxyribonudease
activityin crude nuclearrat liverextracts. (A) Data calculated on the basis or extract DNA concentration,(B) Data calculated on the basisof extract protein concentration.Each point represents the mean of 8-10 independentmeasuresobtained from at least 6 animals per group, Vertical lines represents the standard deviation,
was detected. The activity measures ranged close to the mean values: 11.45 nmole/(min × mg DNA) or 1.90 nmole/(min × mg protein). Discussion
The repair of AP sites and O6-m-gua in the liver of Fischer 344 rats displays quite different behaviours throughout the lifetime of the animals. So the mechanisms involved in the control of these activities appear independently regulated in the ageing. It was observed that AP-endo activities are invariably present at high constitutive levels during the lifetime of the animals. Because the AP sites represent a DNA lesion that is widely produced spontaneously and by a considerable number of agents, it is not surprising that the AP-endo activities are expressed like 'housekeeping' gene products in order to assure normal tissue development. By contrast, the O~-MT activity appears to be age-related only when the results are expressed as a function of DNA concentration of extracts. This normalisation of measures seemed more appropriate to us than protein normalisation because the number of cells per gram of tissue can vary greatly among different animals and among the different stages of tissue development. Consequently, higher data variability obtained using protein parameters, made it impossible for us to measure any significant difference in the O~.MT levels, like Woodhead et al. (1985) or Nakatsuru et al, (1989). The O~-MT enhancement observed within the first month of animal life, using DNA normalisation, could be linked to the liver maturity reached with the normal diet after weaning. We also detected an increase in enzyme until the maximal plateau levels, but only a net decrease of repair activity for the oldest rats was observed, indicating a reduced ability of aged animals to counteract a 'mis-instructive' lesion eventually produced on DNA. The modulation of O6.AT activity accords with findings described in rats by Likhachev et al. (1985) and with similar observations obtained in mice by Nakatsuru et al. (1989). From these results it appears that the susceptibility of rat liver to alk'ylating agents could be higher in the youngest and in the oldest animals. Nevertheless, extrapolations of the age-associated spon-
taneous tumour incidence need further assessment. Moreover, the inducibility of O6-AT by a number of different agents may suggest an important role of this repair activity in ageing. In fact O6-AT has been reported to be induced, in aerobic conditions, by X rays, y rays or bleomycin, indicating a possible involvement of "OH radicals (Margison et al., 1985; Sehmerold and Spath, 1986). This enzyme modulation could represent a nuclear marker for the cumulative damage by active oxygen, and O6-AT could be important in the cell defence perhaps as radical scavenger (yon Hofe and Kennedy, 1988). On the other hand the age-related accumulation of aikyl damages in the DNA o," animals (Ames, 1989) could be crucial in old rats. The less effective O6-m-gua repair could contribute, besides the induction of harmful mutations, to the modification of 5-methyicytosine patterns, as observed in aging (Ono et al., 1989; Drinkwater et al., 1989), producing possible alterations of gene expression. Acknowledgement This work was partially supported by the Italian Association for Cancer Research (AIRC).
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