[CANCER RESEARCH 50, 2759-2764, May I. 1990] Accumulation of 06-Methylguanine in Human Blood Leukocyte DNA during Exposure to Procarbazine and Its Relationships with Dose and Repair1 Yassilis L. Souliotis, Stella Kaila, Viki A. Boussiotis, Gerasimos A. Pangalis, and Soterios A. Kyrtopoulos2 Program of Chemical Carcinogenesis, National Hellenic Research Foundation, Institute of Biological Research, 48 Vassileos Constantinou Avenue ¡V.L. S., S. K,, S, A, K.], and Lymphoma Clinic, Hematology Unit, University of Athens School of Medicine [V. A. B., G. A. P], Athens 116 35, Greece ABSTRACT O'-Methylguanine was measured by a competitive repair assay in blood leukocyte DNA of seven patients with Hodgkin's or non-Hodgkin's lymphoma during therapeutic exposure to procarbazine involving three daily p.o. doses (50 mg each) for 10 days (corresponding to 2.1 mg/kg/ day for a 70-kg human). Adduci accumulation was observed in all seven cases, reaching levels up to 0.28 fmol/fig of DNA (0.45 ¿imol/mol of guanine). In one individual, maximal levels of adduct were reached after 7 days of exposure, followed by a steady decline, whereas in all other individuals continuous accumulation was observed throughout the expo sure period. In four individuals for which data were available for Day 11 (12 to 16 h after the final intake of procarbazine), decreased amounts of 06-methylguanine were observed relative to the last previous measure ments. The accumulation of 06-methylguanine was linearly correlated (/' < 0.01) with the cumulative dose of procarbazine, with a slope of 0.011 fmol of 06-methylguanine/fig of DNA per mg/kg of body weight or 2.68 x 10"" fmol of 0" methylguanine DNA per mg/m2. (Two h after the administration of single p.o. doses of 1 to 10 mg/kg of procarbazine to rats, O'-methylguanine formation in leukocyte DNA was just under half that in liver DNA and showed a linear relationship with dose with a slope of 0.017 fmol/Ã-Ã-g of DNA per mg/kg of body weight or 5.67 x 10"" fmol of 06-methylguanine/#<g of DNA per mg/m2. A negative correlation (P < 0.05) between the rate of accumulation of 06-methylguanine in different individuals and lymphocyte O'-alkylguanine-DNA alkyltransferase (ACT) was observed, demonstrating a probable protective effect of ACT against the accumulation of 06-methylguanine during exposure to meth- ylating agents. This observation supports the suggestion of a possible role of procarbazine-induced O'-methylguanine in the pathogenesis of acute nonlymphocytic leukemia appearing after treatment with chemo- therapeutic protocols which include procarbazine, based on the finding of low lymphocyte ACT levels in patients with such therapy-related neoplastic disease (Sagher et al., Cancer Res., 48: 3084-3089, 1988). Lymphocyte ACT levels were mainly in the range of 5 to 10 fmol/MKof DNA and showed no consistent variation during procarbazine exposure. INTRODUCTION PCZ3 («-isopropyl-a-(2-methylhydrazino)-p-toIuamide hy- drochloride) is a cytotoxic drug used with considerable success in the chemotherapy of a number of human neoplasias, partic ularly Hodgkin's and non-Hodgkin's lymphomas (I, 2). The use, however, of combination chemotherapy protocols which include PCZ is also associated with an increased frequency of secondary acute nonlymphocytic leukemia (3-5). Although this carcinogenic effect cannot be attributed specifically to PCZ, it is possible that this drug, which is a known animal carcinogen (6-8), may contribute to it. Received 9/18/89: revised 1/4/90. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1This work was supported by grants awarded to S. A. K. by the European Economic Community (Contract EV4V-0062) and the International Agency for Research on Cancer (Collaborative Research Agreement BRI/89/09). 2To whom requests for reprints should be addressed. 3The abbreviations used are: PCZ. procarbazine: O'-mGua, O'-methylgua- nine: ACT. O'-methylguanine-DNA alkyltransferase: CRA. competitive repair assay; HPLC. high-performance liquid chromatography; DTT, dithiothreitol: PBS, phosphate-buffered saline: SDS. sodium dodecyl sulfate: TE buffer, 10 IHM Tris-HCl (pH 8.0):50 mM EDTA. The bioactivation of PCZ involves its initial oxidation into azoprocarbazine (9). This is further metabolized into a mixture of methylazoxyprocarbazine derivatives which can ultimately lead to the generation of methylating or arylating diazonium ions. A separate metabolic pathway probably involving free radicals and resulting in the formation of methane also exists. Following the administration of radiolabeled PCZ to experi mental animals, the formation of methylated adducts in DNA has been demonstrated (10, 11). After p.o. exposure of rats, high levels of methylated adducts have been found in the liver and mammary tissue, while lower levels have been observed in the lung and gastrointestinal tissues. Among the products of the methylation of DNA by the methyldiazonium ion or related intermediates, O6-mGua has been implicated in the mechanism of mutagenesis and carcinogenesis. It is a directly miscoding lesion whose presence in DNA during cell replication can result in the generation of G:A transitions which can, in turn, lead to the activation of oncogenes (12). Furthermore, the accumula tion of O6-mGua in particular types of tissues or cells during experimental carcinogenesis has been shown to correlate with the appearance of cancer (13, 14). It is possible, therefore, that O6-mGua may play a causative role in any carcinogenic activity of PCZ in humans. Evidence supporting the possibility of O6- mGua involvement in the pathogenesis of acute nonlymphocy tic leukemia following therapeutic exposure to PCZ was re cently presented by Sagher et al. ( 15) who have reported that patients with therapy-related neoplasia have lower lymphocyte levels of AGT, the enzyme responsible for repair of O6-mGua. This implies that O6-mGua may accumulate to an increased extent during PCZ exposure in individuals with low AGT and thus result in increased risk of subsequent carcinogenesis. There is evidence that the mechanism of the cytotoxic action of PCZ may also be mediated by the methylating metabolic pathway. A significant recent finding supporting this view is that methylazoxyprocarbazine, an intermediate on this path way, is more cytotoxic than PCZ itself, while benzylazoxypro- carbazine (the corresponding arylating metabolite) lacks cyto toxic activity (16, 17). Although the cytotoxic activity of simple methylating agents has for a long time been attributed to adducts other than O6-mGua, there has been mounting evidence during recent years that the latter may also contribute to cyto- toxicity (18-20). Evidence supporting the involvement of O6- mGua in the cytotoxic (and antineoplastic) activity of PCZ was recently produced by Schold et al. (21) who showed that admin istration of PCZ to mice bearing xenografts of human brain tumors results in greater growth delay of those tumors which are of the mer~ phenotype, i.e., deficient in AGT. In view of the evidence that O6-mGua may contribute to the carcinogenic as well as the cytotoxic activity of PCZ, it seems possible that the measurement of the accumulation of this adduct in humans during therapeutic exposure to PCZ may serve as a useful index of individual biologically significant exposure (22), with possible implications in the development of improved therapeutic protocols. We wish to report here the results of a pilot study in which O6-mGua and AGT were 2759