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1-nitrosourea to study the role of the DNA repair protein O6-alkylguanine-DNA ... of the mice to 1,3-bis(2-chloroethyl)-1-nitrosourea or O6-benzylguanine alone.
Journal of Reproduction and Fertility (2000) 119, 339–346

Role of O6-alkylguanine-DNA alkyltransferase in the resistance of mouse spermatogenic cells to O6-alkylating agents M. J. Thompson1, S. Abdul-Rahman2, T. G. Baker2, J. A. Rafferty3, G. P. Margison3 and M. C. Bibby1 1

Clinical Oncology Unit and 2Department of Biomedical Sciences, University of Bradford, Bradford BD7 1DP, UK; and CRC Department of Carcinogenesis, Paterson Institute for Cancer Research, Christie Hospital (NHS) Trust, Wilmslow Road, Manchester M20 4BX, UK

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The O6-alkylguanine-DNA alkyltransferase inactivator O6-benzylguanine was administered to BALB/c mice either alone or before exposure to 1,3-bis(2-chloroethyl)1-nitrosourea to study the role of the DNA repair protein O6-alkylguanine-DNA alkyltransferase in the protection of the testis against anti-cancer O6-alkylating agents. Exposure of the mice to 1,3-bis(2-chloroethyl)-1-nitrosourea or O6-benzylguanine alone did not produce any marked testicular toxicity at the times studied. Testicular O6-alkylguanine-DNA alkyltransferase concentrations were assayed between 0 and 240 min after O6-benzylguanine treatment and were shown to be > 95% depleted 15 min after treatment with O6-benzylguanine and remained at > 95% at all the times assayed. Histological examination, the reduction in testicular mass and the induction of spermatogenic cell apoptosis showed that this depletion significantly potentiated 1,3-bis(2-chloroethyl)-1-nitrosourea-induced testicular damage after treatment. Major histological damage was apparent 42 days after treatment, demonstrating that the stem spermatogonia were significantly affected by the combination. These results demonstrate that O6-alkylguanine-DNA alkyltransferase plays a significant role in protecting the spermatogenic cells from damage caused by DNA alkylation and indicate that the observed toxicity may result from damage to stem spermatogonia.

Introduction The response of the seminiferous epithelium towards chemotherapeutic treatment has been examined using various end-points after exposure to a number of different cytotoxic compounds (Meistrich et al., 1982; Meistrich 1986; Nakagawa et al., 1997; Cai et al., 1997). DNA repair processes involved in the response of the seminiferous epithelium to cytotoxic agents have not been examined extensively, apart from the repair enzymes involved in meiotic recombination (Baker et al., 1996; Edelmann et al., 1996; Anderson et al., 1999). Infertility resulting from chemotherapy remains a problem and, therefore, it is important to begin to identify DNA repair processes that may play a significant role in protecting the spermatogenic stem cells. This knowledge may lead to the identification of patients most at risk of chemotherapy-induced toxicity and reduced fertility. The aim of cancer treatment, including radiotherapy and anti-tumour drug therapy, is to induce tumour regression with the eventual aim of a complete cure. This regression is often dependent upon the production of sufficient DNA damage to induce the cells to undergo cell death. Apoptosis is one mechanism that leads to cell death (Lotem and Sachs, Revised manuscript received 7 January 2000.

1993; Ohmori et al., 1993; Fischer et al., 1994). The chloroethylnitrosoureas and related methylating agents are an important class of anti-tumour drugs that are used to treat a number of tumour types, including brain tumours, Hodgkin’s disease, lymphoma, myeloma and skin cancer (Tew et al., 1995). The cytotoxic action of these compounds is probably due to their ability to alkylate DNA. O6-alkylguanine (O6-alkG) is thought to be the principal cytotoxic, mutagenic, carcinogenic and clastogenic base modification introduced into DNA by these agents, hereafter called the O6-alkylating agents (Meikrantz et al., 1998). The specific spermatogenic cells that undergo apoptosis, either after chemotherapy (Nakagawa et al., 1997; Cai et al., 1997) or exposure to GnRH antagonists (Brinkworth et al., 1995) have been determined. Cells are able to repair DNA damage induced by the O6-alkylating agents, and the major pathway for this is O6-alkylguanine-DNA alkyltransferase (AGT) (D’Incalci et al., 1988; Pegg, 1990; Margison et al., 1996). This pathway acts by the stoichiometric transfer of alkyl moieties to AGT, resulting in the auto-inactivation of the repair protein, and plays an important role in the protection of cells from the cytotoxic and mutagenic effects of O6-alkylating agents (Brent, 1985; Ludlum, 1990; Pegg and Byers, 1992; Nakatsuru et al., 1993). Depletion of AGT concentrations increases the cytotoxicity

© 2000 Journals of Reproduction and Fertility Ltd 0022–4251/2000

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of O6-alkylating agents (Dolan et al., 1985, 1990; Gerson et al., 1988; Mitchell et al., 1992) and, therefore, could be exploited clinically to potentiate the effects of drugs such as 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) against otherwise resistant tumours. O6-benzylguanine (BG) is an example of an AGT inhibitor and has been found to be active at micromolar concentrations (Dolan et al., 1990; Baer et al., 1993; Pegg et al., 1993). BG can potentiate the anti-tumour activity of agents such as BCNU by inhibiting AGT in tumour cells. On the basis of this finding, the combination of BG and BCNU is undergoing phase I clinical trials (Margison et al., 1996; Hickson et al., 1996). However, the effect of the combination of BG and chloroethylating agents on normal tissues has not been addressed fully and is clinically important, since exacerbation of normal tissue toxicity may pose a major dose-limiting problem. One of the central mechanisms protecting the testis from cytotoxic damage due to lesions such as O6-alkylguanine may operate via AGT. Therefore, in the present study mouse seminiferous epithelium was used to study the testicular depletion of AGT activity induced by BG in vivo and to examine the contribution this repair process makes towards protecting the spermatogenic cells, and in particular the stem spermatogonia, from prolonged toxicity induced by alkylating agents.

Materials and Methods The male BALB/c mice (B and K, Hull) were 8 weeks old when treatment commenced. Animals were allowed food and water ad libitum and all work was carried out under a Home Office licence. All drug doses were administered at 0.1 ml per 10 g body weight. BCNU was injected at 10 mg kg−1 i.p. in physiological saline. BG was suspended in 10% (v/v) cremophor EL−saline and administered i.p. at 60 mg kg−1, 2 h before BCNU administration (Dolan et al., 1990, 1994). For histological examination, mice (n = 4–5) were treated with both BCNU and BG and killed 32, 42, 52 and 63 days after treatment, these time points cover histologically detectable damage and recovery (Thompson et al., 1996a). Control animals were treated with solvent vehicle only. Testes were removed, weighed and fixed in Bouin’s fixative. After dehydration, tissues were embedded in paraffin wax for light microscopy and sections were cut and stained with haematoxylin and eosin. For the pharmacokinetics, animals (n = 3) were treated with BCNU alone and in combination with BG as above and samples were taken at 2, 5, 15, 30, 60 and 120 min. For the pharmacokinetic analysis of BCNU concentrations in the testis, a procedure adapted from Yeager et al. (1984) was used. BCNU was extracted by homogenizing the preweighed tissues 1:20 in acetate buffer (pH 4.0, 0.05 mol l−1 at 4οC). Diethyl ether was then added and the mixture was centrifuged for 5 min at 7000 g. The upper layer was transferred into a new vial, evaporated to a dry residue at 35οC and then resuspended in 75 µl mobile phase (32.5% (v/v) acetonitrile and 0.1% (v/v) glacial acetic acid in distilled water). After resuspension, 65 µl of sample was injected into

the HPLC in which BCNU was measured at 229 nm using a reversed phase HPLC (Yeager et al., 1984). In addition, calibration standards were set up using 1−5 µg BCNU ml−1 in drug-free plasma and the same extraction procedure as above. Extraction efficiency was calculated by comparison with a 100% standard of BCNU in saline. The required pharmacokinetic parameters were calculated using standard methods, including the first order elimination constant (Kel), the half life (t1/2) and the area under the curve (AUC), which is a measure of BCNU drug exposure. For the determination of AGT activity, animals (n = 3) were treated with BCNU alone and in combination with BG as above and killed by cervical dislocation at 0, 15, 60, 120 and 240 min after treatment. Tissue samples were frozen rapidly in liquid nitrogen and stored at –80οC before assay. The assay of AGT activity was carried out in a similar manner to that described by Lee et al. (1991, 1992). Briefly, tissue extracts were prepared at 4οC by sonication in buffer I (50 mmol Tris–HCl l−1, 3 mmol DL-dithiothreitol l−1 and 1 mmol EDTA l−1, pH 8.3) plus 0.5 µl leupeptin ml−1 with 10 µl phenylmethylsulphonyl fluoride added after sonication. After centrifugation, the supernatant was stored at –20οC until use. For the assay, triplicate aliquots of the extract were incubated with [3H]-methylated DNA at 37οC for 30 min. After incubation, the DNA substrate was hydrolysed in 1 mol perchloric acid l−1 by heating at 75οC for 50 min. The samples were then centrifuged at 20 000 g for 10 min and washed in 1 mol perchloric acid l−1 before resuspending them in 0.01 mol sodium hydroxide l−1 and dissolving them in aqueous scintillation fluid. Radioactivity was determined by scintillation counting. Protein was estimated using the Bradford (1976) method with BSA as the standard. AGT activity was calculated from the linear part of the protein dependence curve and expressed as femtomoles of methyl groups transferred to protein per milligram of total protein in the extract. Animals (n = 5) were treated as above and killed 1, 24 and 48 h after treatment to identify which cell types were undergoing cell death in response to treatment. Testes were removed, fixed in formalin, embedded in paraffin wax and 5 µm sections were cut. The TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling) protocol was used with an ApopTag–peroxidase kit (Oncor, Gaithersburg, MD) to label apoptotic cells. Briefly, sections were deparaffinized and rehydrated, digested with 20 µg proteinase K ml−1, treated with 3% (v/v) hydrogen peroxidase in PBS to quench endogenous peroxidase activity before incubation with terminal deoxynucleotidyl transferase (TdT) and digoxigenin-11-dUTP at 37°C for 1 h. After this time, the reaction was stopped with stop-wash buffer. Sections were treated with anti-digoxigenin peroxidase before being stained with diaminobenzidine (DAB) substrate, counterstained with methyl green, dehydrated and mounted for examination. Negative staining was performed with distilled water replacing the TdT enzyme. Apoptosis was determined for both combined spermatogonia−spermatocytes and spermatids in 100 cross-sections from different regions of the testis for each mouse scored. Tubules were staged according to criteria set out by Russell et al. (1990) and the results were presented in a manner similar to those of Cai et al. (1997).

Inactivation of testicular alkyltransferase

Results The mean AGT activity in the testis of healthy 10-week-old BALB/c mice (Fig. 1) was 96 ⫾ 22.6 fm mg–1. This activity decreased rapidly after treatment with 60 mg BG kg−1 (Fig. 1). After 15 min, the AGT activity had fallen to 7.5% of the pre-treatment activity (7.07 ⫾ 1.85 fm per mg total cellular protein) and, by 60 min after BG treatment, the activity had fallen further to 4.6% of the pre-treatment value (4.37 ⫾ 0.94 fm mg–1 total cellular protein). After 2 and 4 h, the AGT concentrations were < 2 fm mg–1 total cellular protein, which represents the limit of detection of this assay and substrate. Pharmacokinetic analysis of BCNU concentrations within the testis (Fig. 2) demonstrated that concentrations were highest (4.54 µg ml−1) immediately after treatment and, thereafter, decreased rapidly so that, after 2 h, they had decreased by > 96% to 0.18 µg ml−1. Experiments carried out in triplicate demonstrated that BCNU concentrations were unaffected by pre-treatment with BG. No significant toxic effect was associated with treatment with BG alone: all spermatogenic cell types were present and all tubules were easily staged (Fig. 3). However, some damage was observed in testicular tissue 32 days after BCNU treatment alone (10 mg kg−1) (Fig. 3). The non-spermatogenic cells were unaffected by either treatment alone. The combination of 60 mg BG kg−1 followed 2 h later by 10 mg BCNU kg−1 was much more toxic than either agent given alone. Histologically, all spermatogenic and nonspermatogenic cell types remained unaffected (Fig. 3) 32 days after treatment. At 42 days after treatment, a marked

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Fig. 1. Mean ⫾ SEM testicular O6-alkylguanine-DNA alkyltransferase (AGT) activity (fm mg−1) over time (0–4 h) in BALB/c mice (n = 3) after treatment with 60 mg O6-benzylguanine kg−1. BCNU concentration (µg ml–1)

The spermatid micronucleus assay, based on that of Tates et al. (1984), was used as a measure of clastogenic cellular damage induced by an O6-alkylating agent during BG depletion of AGT. This assay was designed to cover both non-lethal damage and that sufficient in nature to induce lethal cell death processes. Animals (n = 4) were treated as above and killed after 1, 24 and 48 h. Briefly, the testes, without tunica albuginea, were removed and incubated with collagenase (0.5 mg ml−1) and the tubules were allowed to sediment, which facilitated removal of the supernatant containing the interstitial cells. The isolated tubules were then incubated in trypsin (0.5 mg ml−1) before the cell aggregates were gently broken apart by pipetting. The resulting cell suspension was then filtered through a 80 µm filter, rinsed and re-suspended in RPMI tissue culture medium. Slides were prepared by placing three drops of cell suspension onto a slide and then adding Helly’s fixative 15 min later. After 60 min, the slides were transferred to 70% ethanol before staining with periodic acid−Schiff and counterstained with 15% Mayer’s haemalum for 10 min. For each animal, 1000 stage I–III Golgi-phase spermatids were scored. The statistical significance of the results was determined using ANOVA and Tukey’s HSD for multiple pairwise comparisons, using the SPSS statistical PC package. Differences of P < 0.05 were considered significant.

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40 120 60 80 100 Time (min) after treatment Fig. 2. Mean ⫾ SEM concentration of 1,3-bis(2-chloroethyl)-1nitrosourea (BCNU) (µg ml−1) in the testis of BALB/c mice (n = 3) over time after treatment with 10 mg BCNU kg−1 i.p.

and widespread difference in the appearance of the seminiferous epithelium (Fig. 3) was observed in slides from animals treated with both BCNU and BG compared with those given either treatment alone. Morphologically, the tubules of animals treated with both BCNU and BG were frequently shrunken and difficult to classify owing to the loss of cells and this morphology corresponded to a significant reduction in testis mass after 42 days (Fig. 4) but histologically, the non-spermatogenic cells appeared unaffected (Fig. 3). Recovery appeared to be complete at 63 days after treatment, and this conclusion was confirmed histologically (data not shown) and by the testes mass values (Fig. 4). When the apoptotic cells were examined, there was no significant change in the number of apoptotic cells in the testes from mice treated with BG only. There was an increase in the number of apoptotic stages I–IV spermatogonia− spermatocytes in the BCNU-treated tissue (P < 0.05). In the testes treated with both BG and BCNU, there was a significant increase in the number of apoptotic stages I–VIII spermatogonia−spermatocytes (P < 0.05) (Fig. 5). All other spermatogenic stages were unaffected by the treatments. The increase in the number of apoptotic cells was surprisingly rapid, and significant increases were observed after 1 h,

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Fig. 3. Typical appearance of seminiferous tubules from BALB/c mice after various treatments. (a) Control (stages II–III) tubule. (b) Tubule (stages VI–VII) 32 days after exposure to 60 mg O6-benzylguanine (BG) kg−1. (c) Tubule (stage could not be determined) 32 days after exposure to 10 mg 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) kg−1. (d) Tubule (stages X–XI) 42 days after exposure to 10 mg BCNU kg−1. Tubules 32 days (stage of central tubule could not be determined owing to cell loss but surrounding tubules at stages X–I) (e) and 42 days (f) after exposure (stage of tubule could not be determined owing to extensive cell loss) to combined BG (60 mg kg−1) and BCNU (10 mg kg−1). Staining was with haematoxylin and eosin. Scale bars represent 25 µm.

reaching a maximum after 24 h. The 12 h time point at which testicular apoptosis reached peak values in rats (Cai et al., 1997) was not examined in the present study, but the results presented here do indicate which spermatogenic cells were induced to undergo apoptosis, as part of the larger study of the role of testicular AGT. BG had no effect on induction of micronuclei in early

spermatids (Fig. 6) over the period of time examined (1−18 days after treatment). Treatment with BCNU alone led to a significant increase in induction of micronuclei (P < 0.05) 18 days after treatment. When induction of micronuclei was examined after treatment with both BG and BCNU, no significant increase was found between day 1 and day 3. The presence of micronuclei could not be assessed at day 18

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owing to the absence of early spermatids, presumably as a result of the cytotoxic nature of the BG−BCNU combination.

Discussion 6

The activity of O -alkylguanine-DNA alkyltransferase in the testis was found to be comparable to that observed in other cells and tissues, such as testicular tumour cells (Walker et al., 1992), lymphocytes and metastatic melanoma (Lee et al., 1992) and other normal mouse tissues (Dolan et al., 1990). In the present study, the activity of AGT was reduced by > 90% within 15 min of treatment with 60 mg BG kg−1 and, after 2 h, had decreased to below the level of detection in the assay. The kinetics and degree of inhibition found in the testis after 60 mg BG kg−1 is similar to that reported for inactivation in other tissues, such as the liver, bone marrow (Bibby et al., 1999) and tumour tissue grown as xenografts (Felker et al., 1993), and parallels the depletion of AGT in human lymphocytes when treated with the methylating agent CB10-277 in vivo (Lee et al., 1992). In the absence of an alkylating agent, this inhibition does not produce any detectable toxicity in the testis, in agreement with other studies of the effect of administration of BG on testis and other tissues (Bronstein et al., 1992; Thompson et al., 1996a; Dolan et al., 1998; Friedman et al., 1998). Recovery of AGT activity in tissues is slow, taking between 16 and 72 h to reach

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pre-treatment activity (Dolan et al., 1990). Pharmacokinetically, BCNU concentrations were found to decrease rapidly in the testis, with concentrations dropping > 90% in the first 2 h. This finding indicates that when BCNU concentrations are at their highest, AGT concentrations are at their lowest, allowing maximum damage to occur. Over a longer period, the histology of the seminiferous epithelium was not significantly affected by BCNU alone. After 32 or 42 days, the normal profile of the spermatogenic tissue was present with no discernible abnormalities. BCNU alone did cause some damage, as shown by the increased number of micronuclei 18 days after treatment and the increase in apoptotic spermatogonia−spermatocytes at stages I–IV. The observation that this damage did not significantly disturb the histology of the seminiferous epithelium or produce a decreased testis mass at the later times studied indicates that the stem spermatogonia were not significantly affected. Significant histological damage and reduction in testis mass were noted after 42 days. The spermatogenic cycle in mice has a duration of about 35 days (Clermont, 1972) and, therefore, the damage observed 42 days after treatment (Meistrich, 1986) indicates that the stem spermatogonia were affected by the combination treatment, leading to histologically apparent damage in the progeny of these cells later than 35 days after treatment. The extent of any potential cellular recovery after exposure to BG and BCNU would be determined by the degree to which the stem spermatogonia had been damaged. Permanent azoospermia would only occur if these cells have been damaged irreversibly, and so are unable to repopulate the seminiferous tubules to restore fertility to pre-treatment concentrations. The appearance of the histological damage 42 days after treatment indicates that the adducts initially may be non-lethal to the stem cells. It is more likely that the stem cells sustain significant damage, but that their response (arrest, repair and apoptosis) is realised only when they re-enter mitosis to replace lost differentiating spermatogonia. After the combined BG and BCNU treatment, there were insufficient early spermatids remaining at day 18 in which to score micronuclei. This finding indicates that, in agreement with Meistrich et al. (1982), early spermatogonia were significantly affected by the cytotoxic effects of O6-alkG (combined with a depletion of AGT-based repair in the present study) at the point of treatment. Specifically, this effect occurs on spermatogenic cells between late A spermatogonia and pre-pachytene spermatocytes. The apoptosis data and those of Cai et al. (1997) support this finding, demonstrating that, up to 48 h after treatment, it is the spermatogonia−spermatocytes at stages I–VIII that are affected by the O6-alkG adduct. The present results indicate that the spermatogenic cells are normally protected by the expression of concentrations of AGT high enough to remove most of the BCNU-induced damage. Thus, when BCNU is given alone, the available AGT can protect against the cytotoxic effects of O6-alkG, and the overall cellularity of the testis is not significantly disturbed. The inactivation of this mechanism by BG reduces the capacity of the testis for repair and thus potentiates the biological effects of the DNA damage in a similar manner to

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Fig. 5. Apoptosis induced (mean ⫾ SEM number of apoptotic cells per 100 tubules) in the seminiferous epithelium of BALB/c mice 1, 24 and 48 h after treatment with 10 mg 1,3-bis(2-chloroethyl)-1-nitrosourea kg−1 (䊏), 60 mg O6benzylguanine kg−1, alone (䊉) or in combination (⫻). Spermatogonia−spermatocytes at stages I−IV (a), V−VIII (b) and IX−XII (c); spermatids at stages I−IV (d) and V−VIII (e). 䉱, Control. *Significantly different from control (P < 0.05).

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that observed in bone marrow treated with nitrosoureas (Matthew et al., 1994; Fairbairn et al., 1995; Thompson et al., 1996b). These biological effects produce an initial increase in spermatogenic apoptosis resulting in greater cell loss from the seminiferous epithelium later after treatment. While the results of the present study demonstrate that early spermatogonia were damaged, recovery occurred, with an increase in the number of spermatogenic cells after the nadir observed 42 and 53 days after treatment. This observation indicates that, in the present study, sufficient stem spermatogonia remain after treatment to ensure an effective and rapid spermatogenic repopulation and a return to fertility.

BCOd3 Od18 BCd18 BCOd18

Treatment type and time Fig. 6. Number (± SEM) of micronuclei in the early spermatids of BALB/c mice 1, 3 and 18 days after treatment with 10 mg 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) kg−1, 60 mg O6-benzylguanine

(BG) kg−1, alone or in combination. Con: control; Od1/Od18: BG alone after 1 or 18 days; BCd1, BCd3 and BCd18: BCNU after 1, 3 and 18 days, respectively; BCOd1, BCOd3 and BCOd18: BCNU and BG in combination after 1, 3 and 18 days, respectively. *Significantly different from control (P < 0.05).

Inactivation of testicular alkyltransferase Cells possess various mechanisms by which they can repair damaged DNA, such as the nucleotide excision repair (NER) or base excision repair pathways (Myles and Sancar, 1989). A role for NER in the repair of some alkylation damage has been suggested by Lukash et al. (1991) and, even at high expression, AGT does not always confer resistance in relation to its activity (Walker et al., 1992). The present results indicate that, in this model, AGT plays a significant role in protecting the spermatogenic tissues. In conclusion, testicular AGT activity was rapidly reduced by treatment with BG. The combination of BG with doses of BCNU that are not cytotoxic when adminstered alone potentiates the toxicity of this nitrosourea, particularly against the stem spermatogonia, and demonstrates the importance of AGT in protecting the testis from the O6-alkylguanine base modification. The authors wish to acknowledge the invaluable assistance and advice of Paul Loadman with the pharmacokinetic analyses.

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