Duplex sequencing provides detailed characterization of mutation frequencies and spectra in the bone marrow of MutaMouse males exposed to procarbazine hydrochloride.

Mutagenicity testing is an essential component of health safety assessment. Duplex Sequencing (DS), an emerging high-accuracy DNA sequencing technology, may provide substantial advantages over conventional mutagenicity assays. DS could be used to eliminate reliance on standalone reporter assays and provide mechanistic information alongside mutation frequency (MF) data. However, the performance of DS must be thoroughly assessed before it can be routinely implemented for standard testing. We used DS to study spontaneous and procarbazine (PRC)-induced mutations in the bone marrow (BM) of MutaMouse males across a panel of 20 diverse genomic targets. Mice were exposed to 0, 6.25, 12.5, or 25 mg/kg-bw/day for 28 days by oral gavage and BM sampled 42 days post-exposure. Results were compared with those obtained using the conventional lacZ viral plaque assay on the same samples. DS detected significant increases in mutation frequencies and changes to mutation spectra at all PRC doses. Low intra-group variability within DS samples allowed for detection of increases at lower doses than the lacZ assay. While the lacZ assay initially yielded a higher fold-change in mutant frequency than DS, inclusion of clonal mutations in DS mutation frequencies reduced this discrepancy. Power analyses suggested that three animals per dose group and 500 million duplex base pairs per sample is sufficient to detect a 1.5-fold increase in mutations with > 80% power. Overall, we demonstrate several advantages of DS over classical mutagenicity assays and provide data to support efforts to identify optimal study designs for the application of DS as a regulatory test.

Introduction of Z-GP scaffold into procarbazine reduces spermatoxicity and myelosuppression.

Incorporation of carbobenzoxy-glycylprolyl (Z-GP) to either α or ß position of the hydrazine moiety in procarbazine (Pcb) has been carried on in 5-steps process. The overall yield was 32.7%. The new entity Z-GP-Pcb was confirmed targeting to fibroblast activation protein-α (FAPα). Z-GP-Pcb may be hydrolyzed by either isolated rhFAPα or tumor homogenate. It was shown far less cytotoxicity against NCI-H460 cell line than Pcb. Z-GP-Pcb was displayed the potency to reduce spermatoxcity in H22-bearing mice. The mechanism may be ascribed to the blockade of dehydrogenation by α-glycerolphosphate dehydrogenase. This candidate was further proved equal antitumor activity to Pcb. However, the introduction of Z-GP scaffold decreased myelosuppression. All the evidences support that Z-GP-Pcb is a better antitumor agent than Pcb.

Spectrum of Pig-a mutations in T lymphocytes of rats treated with procarbazine.

Procarbazine is a primary component of antineoplastic combination chemotherapy often used for the treatment of Hodgkin's lymphoma. It is believed that cytostatic and cytotoxic properties of procarbazine are mediated via its interaction with genomic DNA. Procarbazine is a carcinogen in animal models; it is classified as Group 2A compound by IARC. Also it is known as an in vitro and in vivo mutagen and genotoxicant. However, the molecular mechanism by which procarbazine induces mutations is not thoroughly understood and the spectrum of procarbazine-induced in vivo mutations is described insufficiently. We employed flow cytometry-based erythrocyte and T lymphocyte assays in order to quantify the frequencies of cells deficient in glycosylphosphatidyl inositol-anchored surface markers CD59 and CD48 (presumed mutants in the endogenous X-linked Pig-a gene) in rats. The rats were treated once daily with 100 mg/kg procarbazine HCl for 3 days. In addition, we sorted mutant-phenotype spleen T cells and immediately analysed their Pig-a gene using next generation sequencing of dual-indexed multiplex libraries and error-correcting data filtering. More than 100-fold increase in the frequencies of CD59-deficient RBCs was observed at Day 29 after the last administration, and a 10-fold increase in the frequency of CD48-deficient T cells was observed at Days 45 to 50. Sequencing revealed that, in T cells from procarbazine-treated rats, mutations in the Pig-a gene occurred predominantly at A:T basepairs when A was located on the non-transcribed DNA strand. A→T transversion was the most common mutation. Our results suggest that, at least for the transcribed X-linked Pig-a gene, in vivo methyl guanine adducts are not the major contributors to mutations induced by procarbazine.

Antigenotoxic effects of resveratrol: assessment of in vitro and in vivo response.

Experiments were performed to evaluate the in vitro and in vivo dose response for antigenotoxic effects of resveratrol (RES). For the in vitro study, HL-60 cells were co-treated with the test genotoxin and three concentrations of RES. Thereafter, genotoxic effects were assessed in the cytokinesis-block micronucleus test. Results of the in vitro experiments using genotoxins nitroquinoline-1-oxide (NQO) and mitomycin C (MMC) showed maximum inhibition of genotoxicity with the lowest test concentration of RES. The mouse bone marrow micronucleus assay was used for evaluating the in vivo antigenotoxic effects of RES against genotoxins diepoxybutane (DEB), MMC, methyl methanesulfonate and procarbazine (PCB). The experimental animals received RES pre-treatment by gavage 30min, 24 and 48h before injecting the genotoxin intraperitoneally. The in vivo studies demonstrated efficacy of the lowest test dose of RES for exerting maximum protection against chromosomal damage induced by all four genotoxins. The antigenotoxic effect observed with 6.25mg/kg RES was significantly higher than that of 100mg/kg RES against PCB and DEB. In conclusion, the findings from the present study indicate that lower test concentrations/doses of RES are more effective in exerting antigenotoxic effects.

Treatment of large low-grade oligodendroglial tumors with upfront procarbazine, lomustine, and vincristine chemotherapy with long follow-up: a retrospective cohort study with growth kinetics.

We treated patients with newly diagnosed and large low-grade oligodendroglial tumors with upfront procarbazine, CCNU and vincristine (PCV) in order to delay radiotherapy. Patients were treated with PCV for a maximum of 6 cycles. The response to treatment was defined according to the RANO criteria; in addition change over time of mean tumor diameters (growth kinetics) was calculated. Thirty-two patients were treated between 1998 and 2006, 18 of which were diagnosed with 1p/19q co-deleted tumors. Median follow-up duration was 8 years (range 0.5-13 years). The median overall survival (mOS) was 120 months and the median progression-free survival (mPFS) was 46 months. Growth kinetics showed an ongoing decrease of the mean tumor diameter after completion of chemotherapy, during a median time of 35 months, but an increase of the mean tumor diameter did not herald progression as detected by RANO criteria. 1p/19q co-deletion was associated with a significant increase in OS (mOS 83 months versus not reached for codeleted tumors; p = 0.003)) and PFS (mPFS 35 months versus 67 months for codeleted tumors; p = 0.024). Patients with combined 1p/19q loss had a 10 year PFS of 34 % and the radiotherapy in these patients was postponed for a median period of more than 6 years. This long-term follow-up study indicates that upfront PCV chemotherapy is associated with long PFS and OS and delays radiotherapy for a considerable period of time in patients with low-grade oligodendroglial tumors, in particular with combined 1p/19q loss.

Protective effects of common anthocyanidins against genotoxic damage induced by chemotherapeutic drugs in mice.

Experiments were performed to assess in mice the inhibitory effects of the anthocyanidins cyanidin, delphinidin, malvidin, and pelargonidin on genotoxic damage induced by the anticancer drugs cyclophosphamide, procarbazine, and cisplatin. Each anthocyanidin was administered 30 min before injecting the drug, and genotoxicity was assessed by measuring micronucleated polychromatic erythrocytes in bone marrow cells. In addition, we monitored the effect of anthocyanidins on apoptosis induced by cyclophosphamide and procarbazine. The results showed significant protective effects of cyanidin, delphinidin, malvidin, and pelargonidin against DNA damage induced by cyclophosphamide. With delphinidin and malvidin, a biphasic dose-response was observed for protection against cyclophosphamide. Dose-related reduction of genotoxicity was observed with pelargonidin against procarbazine. However with cyanidin, the medium dose of 2 mg/kg showed maximum protection against procarbazine. Cyanidin and pelargonidin significantly reduced the chromosomal damage induced by cisplatin. Furthermore, pre-treatment with these anthocyanidins reduced the level of apoptosis induced by cyclophosphamide and procarbazine. In conclusion, this study shows that anthocyanidins can reduce the efficacy of anticancer drugs for inducing DNA damage and apoptosis.

Mutational signatures in T-lymphocytes of rats treated with N-propyl-N-nitrosourea and procarbazine.

We have used whole genome sequencing (WGS) to determine mutational signatures induced in the T-cells of rats treated in vivo with N-propyl-N-nitrosourea (PNU) or procarbazine (PCZ). The signatures from the treated rats were different from the signature of background mutations. The main component of the spontaneous T-cell mutational signature was C➔T transition with all other single base substitutions evenly distributed. The PNU-induced mutational signature showed relatively equal contributions from C➔T and T➔C transitions, and T➔A transversions. The PCZ-induced signature was characterized by T➔C transitions, T➔A and, to a smaller extent, T➔G transversions. C➔G transversions were infrequent in either the PNU or PCZ signatures. WGS not only allowed mutational signature detection, but also measured quantitative responses to mutagen treatment: 10-40× increases in the number of mutations per clone were detected in T-cell clones from treated rats. The overall strand specificity of induced mutations for annotated rat genes was comparable to the strand specificity of mutations determined previously for the endogenous X-linked Pig-a gene. Our results provide valuable reference data for future applications of WGS in safety research and risk assessment.

Pig-a gene mutations in bone marrow granulocytes of procarbazine-treated F344 rats.

It was previously demonstrated that procarbazine (PCZ) is positive in the rat erythrocyte Pig-a gene mutation assay. However, since mammalian erythrocytes lack genomic DNA, it was necessary to analyze nucleated bone-marrow erythroid precursor cells to confirm that PCZ induces mutations in the Pig-a gene (Revollo et al., Environ Mol Mutagen, 2020). In this study, the association between Pig-a mutation and loss of GPI anchors was further strengthened and the genesis of Pig-a mutation in PCZ-dosed rats was evaluated by analyzing bone-marrow granulocytes. Erythrocytes and granulocytes both originate from myeloid progenitor cells, but granulocytes contain DNA throughout their developmental stages. F344 rats were treated with three doses of 150 mg/kg PCZ; 2 weeks later, CD48-deficient mutant phenotype bone-marrow granulocytes (BMGs [CD11b+ ]) were isolated by flow-cytometric sorting. Sequencing data showed that the CD48-deficient mutant phenotype BMGs contained mutations in the Pig-a gene while wild-type BMGs did not. PCZ-induced mutations included missense, nonsense and splice site variants; the majority of mutations were A > T, A > C, and A > G, with the mutated A on the nontranscribed DNA strand. The PCZ-induced mutational analysis in BMGs supports the association between the phenotype measured in the Pig-a assay and mutation in the Pig-a gene. Also, PCZ mutation spectra were similar in bone-marrow erythroids and BMGs, but none of the mutations detected in BMGs were the same as the erythroid precursor cell mutations from the same rats. Thus, mutations induced in the Pig-a assay appear to be induced after commitment of myeloid progenitor cells to either the granulocyte or erythroid pathway.

CD59-deficient bone marrow erythroid cells from rats treated with procarbazine and propyl-nitrosourea have mutations in the Pig-a gene.

Procarbazine (PCZ) and N-propyl-N-nitrosourea (PNU) are rodent mutagens and carcinogens. Both induce GPI-anchored marker-deficient mutant-phenotype red blood cells (RBCs) in the flow cytometry-based rat RBC Pig-a assay. In the present study, we traced the origin of the RBC mutant phenotype by analyzing Pig-a mutations in the precursors of RBCs, bone marrow erythroid cells (BMEs). Rats were exposed to a total of 450 mg/kg PCZ hydrochloride or 300 mg/kg PNU, and bone marrow was collected 2, 7, and 10 weeks later. Using a flow cell sorter, we isolated CD59-deficient mutant-phenotype BMEs from PCZ- and PNU-treated rats and examined their endogenous X-linked Pig-a gene by next generation sequencing. Pig-a mutations consistent with the properties of PCZ and PNU were found in sorted mutant-phenotype BMEs. PCZ induced mainly A > T transversions with the mutated A on the nontranscribed strand of the Pig-a gene, while PNU induced mainly T > A transversions with the mutated T on the nontranscribed strand. The treatment-induced mutations were distributed across the protein coding sequence of the Pig-a gene. The causal relationship between BMEs and RBCs and the agent-specific mutational spectra in CD59-deicient BMEs indicate that the rat RBC Pig-a assay, scoring CD59-deficient mutant-phenotype RBCs in peripheral blood, detects Pig-a gene mutation.

Assessment of the Pig-a, micronucleus, and comet assay endpoints in rats treated by acute or repeated dosing protocols with procarbazine hydrochloride and ethyl carbamate.

The utility and sensitivity of the newly developed flow cytometric Pig-a gene mutation assay have become a great concern recently. In this study, we have examined the feasibility of integrating the Pig-a assay as well as micronucleus and Comet endpoints into acute and subchronic general toxicology studies. Male Sprague-Dawley rats were treated for 3 or 28 consecutive days by oral gavage with procarbazine hydrochloride (PCZ) or ethyl carbamate (EC) up to the maximum tolerated dose. The induction of CD59-negative reticulocytes and erythrocytes, micronucleated reticulocytes in peripheral blood, micronucleated polychromatic erythrocytes in bone marrow, and Comet responses in peripheral blood, liver, kidney, and lung were evaluated at one, two, or more timepoints. Both PCZ and EC produced positive responses at most analyzed timepoints in all tissue types, both with the 3-day and 28-day treatment regimens. Furthermore, comparison of the magnitude of the genotoxicity responses indicated that the micronucleus and Comet endpoints generally produced greater responses with the higher dose, short-term treatments in the 3-day study, while the Pig-a assay responded better to the cumulative effects of the lower dose, but repeated subchronic dosing in the 28-day study. Collectively, these results indicate that integration of several in vivo genotoxicity endpoints into a single routine toxicology study is feasible and that the Pig-a assay may be particularly suitable for integration into subchronic dose studies based on its ability to accumulate the mutations that result from repeated treatments. This characteristic may be especially important for assaying lower doses of relatively weak genotoxicants. Environ. Mol. Mutagen. 60:56-71, 2019. © 2018 Wiley Periodicals, Inc.

Integrated In Vivo Genotoxicity Assessment of Procarbazine Hydrochloride Demonstrates Induction of Pig-a and LacZ Mutations, and Micronuclei, in MutaMouse Hematopoietic Cells.

Procarbazine hydrochloride (PCH) is a DNA-reactive hematopoietic carcinogen with potent and well-characterized clastogenic activity. However, there is a paucity of in vivo mutagenesis data for PCH, and in vitro assays often fail to detect the genotoxic effects of PCH due to the complexity of its metabolic activation. We comprehensively evaluated the in vivo genotoxicity of PCH on hematopoietic cells of male MutaMouse transgenic rodents using a study design that facilitated assessments of micronuclei and Pig-a mutation in circulating erythrocytes, and lacZ mutant frequencies in bone marrow. Mice were orally exposed to PCH (0, 6.25, 12.5, and 25 mg/kg/day) for 28 consecutive days. Blood samples collected 2 days after cessation of treatment exhibited significant dose-related induction of micronuclei in both immature and mature erythrocytes. Bone marrow and blood collected 3 and 70 days after cessation of treatment also showed significantly elevated mutant frequencies in both the lacZ and Pig-a assays even at the lowest dose tested. PCH-induced lacZ and Pig-a (immature and mature erythrocytes) mutant frequencies were highly correlated, with R2 values ≥0.956, with the exception of lacZ vs. Pig-a mutants in mature erythrocytes at the 70-day time point (R2 = 0.902). These results show that PCH is genotoxic in vivo and demonstrate that the complex metabolism and resulting genotoxicity of PCH is best evaluated in intact animal models. Our results further support the concept that multiple biomarkers of genotoxicity, especially hematopoietic cell genotoxicity, can be readily combined into one study provided that adequate attention is given to manifestation times. Environ. Mol. Mutagen. 60:505-512, 2019. © 2018 Her Majesty the Queen in Right of Canada.

Procarbazine--a traditional drug in the treatment of malignant gliomas.

The methylhydrazine derivative Procarbazine (PCZ) as monotherapy or in combination with CCNU and vincristine (PCV) was evaluated in a vast number of clinical trials and is still used in patients with high-grade and low-grade gliomas. The compound is an antineoplastic agent with multiple sites of action. It inhibits incorporation of small DNA precursors, as well as RNA and protein synthesis. PCZ can also directly damage DNA through an alkylation reaction. The drug is not cross-resistant with other mustard-type alkylating agents. As PCZ was in almost all trials used in a combination with CCNU and Vincristin, the efficacy can only be evaluated in the view of the PCV regimen. The published data suggest a role of PCV as a salvage regimen, especially in oligodendroglial tumors; however, well designed studies with high evidence are rare in all entities. This article summarizes the existing data with the goal to define the role of PCZ/PCV in modern neurooncology.

Efficacy of crocin and safranal as protective agents against genotoxic stress induced by gamma radiation, urethane and procarbazine in mice.

Crocin (CRO) and safranal (SAF) are bioactive constituents of saffron (dried stigma of Crocus sativus flower), an expensive spice with medicinal properties. Aqueous extract of saffron is known for its antigenotoxic effect against environmental genotoxins/carcinogens. However, there is need to identify saffron constituents responsible for this antigenotoxic effect. The aim of our investigation was to ascertain the role of CRO and SAF as inhibitors of in vivo genotoxic stress. For this purpose, Swiss albino mice were pretreated with CRO (50-mg/kg body weight (bw))/SAF (0.025- and 0.25-ml/kg bw) by gavage for 2 days. Thereafter, the pretreated mice were exposed to the genotoxic agents: (1) gamma radiation (GR; 2 Gy), (2) urethane (URE; 800 mg/kg) and (3) procarbazine (PCB; 60 mg/kg). In addition, CRO (50 mg/kg) was co-administered with the nitrosation reaction mixture of methylurea (MU; 300-mg/kg bw) + sodium nitrite (15 mg/kg) which can form N-nitroso-N-MU in the stomach. Genotoxic damage was measured by performing the bone marrow micronucleus test. Results obtained demonstrated significant reductions in the incidence of micronucleated polychromatic erythrocytes in the bone marrow of mice pretreated with CRO/SAF before exposure to the above DNA damaging agents, GR, URE and PCB. Co-administration of CRO with the nitrosation reaction mixture led to significant decrease in genotoxicity when compared to nitrosation reaction mixture alone. Histopathological studies revealed that these saffron constituents reduced testicular cell damage induced by the test genotoxins. The cell-free DNA-nicking assay using pBR322 DNA showed significant protective effects of CRO against hydroxyl radical-induced strand breaks.

MOPPEBVCAD chemotherapy with limited and conditioned radiotherapy in advanced Hodgkin's lymphoma: 10-year results, late toxicity, and second tumors.

PURPOSE: MOPPEBVCAD (mechlorethamine, vincristine, procarbazine, prednisone, epidoxirubicin, bleomycin, vinblastine, lomustine, doxorubicin, and vindesine) chemotherapy with limited radiotherapy was devised in 1987 to reduce late toxicity and second tumor incidence while trying to improve effectiveness through increases of dose intensity and dose density. Late results, toxicity, and second tumor incidence were reviewed in all the patients treated. EXPERIMENTAL DESIGN: The drugs of three previous alternating regimens [CAD (lomustine, melphalan, and vindesine), MOPP (mechlorethamine, vincristine, procarbazine, and prednisone), and ABV (doxorubicin, bleomycin, and vinblastine)] were intensified and hybridized, the cumulative dose of mechlorethamine was lowered, and irradiation was delivered to no more than two sites either bulky or partially responding to chemotherapy. RESULTS: A total of 307 previously untreated advanced-stage patients underwent MOPPEBVCAD chemotherapy. Radiotherapy was delivered to 118 of 307 patients (38%). Remission was complete in 290 patients (94%). With a median follow-up of 114 months, 10-year overall, disease-free, and failure-free survival rates were 79%, 84%, and 71%, respectively. Forty-two patients relapsed and 60 died. The causes of death were Hodgkin's lymphoma in 36 patients, second neoplasms in 12, cardiorespiratory diseases in 4, pulmonary diseases in 2, and unknown in 6. Sixteen second tumors (of which nine were myelodysplasia and/or acute leukemia) were diagnosed in all. Outside this series of 307 patients, MOPPEBVCAD obtained complete responses in 12 of 15 relapsed and 9 of 9 refractory patients who had previously been treated with other regimens. CONCLUSIONS: Clinical response and long-term results are very satisfactory, whereas the second tumor incidence was lower than would have been expected with MOPP analogues. Given its response/late toxicity balance, MOPPEBVCAD does not undermine the leading role of ABVD as first-line regimen but can be indicated as a very effective second-line conventional therapy.

Separate mechanisms for procarbazine spermatotoxicity and anticancer activity.

Procarbazine causes dose-dependent decreases in sperm count after a single i.p. injection in (C57BL/6 X DBA/2)F1 male mice. Two antioxidants, N-acetylcysteine and sodium ascorbate, administered with equimolar doses of procarbazine decreased the spermatotoxicity of procarbazine. At the highest doses of procarbazine (400 mg/kg) that caused a 56% decrease in sperm count, equimolar doses of N-acetylcysteine coadministered with procarbazine caused only a 17% decrease in sperm count, and equimolar doses of ascorbate coadministered with procarbazine caused only a 13% decrease in sperm count. Thus, protection against the spermatotoxic effects of procarbazine was demonstrated with either antioxidant. The effect of the antioxidants on the chemotherapeutic efficacy of procarbazine against murine L1210 leukemia was also assessed. Procarbazine at the highest dose (600 mg/kg) increased mean survival time of mice inoculated i.p. with 1 X 10(5) L1210 leukemia cells by 31%. Simultaneous administration of equimolar doses of either N-acetylcysteine or ascorbate given with procarbazine caused no change in the increased mean survival time of tumor-bearing mice. These results indicate a decrease in the toxicity of procarbazine when coadministered with antioxidants, via decreased spermatotoxicity without changing anticancer efficacy. The results also indicate that different mechanisms are involved in the spermatotoxicity and anticancer activity of procarbazine.

Time dependence for the protective effect of androgen from procarbazine-induced damage to rat spermatogenesis.

The protective effects of androgen pretreatment on the procarbazine-induced killing of spermatogonial stem cells in Wistar rats have been investigated. Using testosterone-filled Silastic implants (200 mm2) the degree of protection from four weekly doses of procarbazine (100 mg/kg) was found to be dependent upon the androgen pretreatment time interval as assessed by quantitative histology. No protective effect was seen until rats had received 4 wk of pretreatment with androgen, whereafter protection increased to a maximum (about 20 to 30% of tubule cross-sections exhibiting recovery) after 8 to 12 wk of pretreatment. In contrast, the same level of maximal protection could be obtained by 6 wk of pretreatment using testosterone enanthate, suggesting that differences in protection may be achieved using different modes of androgen administration.

Hormonal protection from procarbazine-induced testicular damage is selective for survival and recovery of stem spermatogonia.

Procarbazine produces long-term sterility in the male by killing stem spermatogonia. The degree and selectivity of protection of stem spermatogonia in rats from procarbazine by pretreatment with steroid hormones were investigated. Male LBNF1 rats were treated for 6 weeks with Silastic implants containing testosterone plus 17 beta-estradiol. The hormone-treated rats and sham-treated controls were given a single injection of graded doses of procarbazine and the hormone implants were removed the next day. Spermatogonial stem cell survival and function, assessed by the repopulation indices and sperm head counts 10 weeks later, showed that stem spermatogonia were protected by testosterone plus 17 beta-estradiol treatment from the toxic effects of procarbazine with a dose-modifying protection factor of about 2.5. In contrast, there was no hormonal protection from the procarbazine-induced killing of differentiating spermatogonia, preleptotene spermatocytes, and spermatocytes in meiotic prophase or from the delay in maturation of round spermatids, assessed 9 days after procarbazine injection by histological or flow cytometric methods. In addition, there was no hormonal protection from the procarbazine-induced decline in body weights and lymphocyte counts, indicating that the gastrointestinal, neurological, and hematological systems were not protected. The specificity of protection indicates that the hormonal protection of the stem spermatogonia is not the result of a systemic or overall testicular decrease in drug delivery, decrease in bioactivation, nor increase in drug detoxification, except possibly within the stem cells themselves. We conclude that the degree of hormonal protection and its specificity would be appropriate for clinical application provided that the mechanism of protection is elucidated and appears applicable to humans.

Partial prevention of procarbazine induced germinal cell aplasia in rats by sequential GnRH antagonist and testosterone administration.

The present study examined the feasibility of using a combination of gonadotropin releasing hormone antagonist (GnRH-A) and testosterone in the prevention of procarbazine induced germinal aplasia. Daily injections of GnRH-A or vehicle were given to adult male rats for 21 days prior to procarbazine (PCB) administration and continued until 2 days after the second of two doses of procarbazine (200 mg/kg i.p.) given 1 week apart. One group of rats receiving GnRH-A and PCB was given s.c. two 5-cm testosterone capsule (TC) implants (inside diameter, 3.5 mm) immediately following the second dose of PCB. Eight weeks after the last PCB treatment, more than 99% of the seminiferous tubular cross-sections of rats receiving PCB alone were devoid of spermatogenic activity. Spermatogenesis in PCB injected animals receiving GnRH-A pretreatment alone was abortive but was partially preserved when exogenous testosterone was given following PCB administration. At 16 weeks, spermatogenesis was absent in all PCB treated animals and was only observed in less than 1% of the tubular cross-sections of the PCB treated rats receiving GnRH-A pretreatment alone. On the other hand, active spermatogenesis was noted in 68% of the tubular cross-sections, and complete spermatogenesis was noted in four of the five PCB treated rats receiving both GnRH-A pretreatment and subsequent TC implantation. At the time of sacrifice, testicular testosterone concentrations in animals receiving TC implants were below 10% of normal levels, while both serum and testicular testosterone content were increased in PCB treated animals with or without GnRH-A pretreatment. Concomitantly, testicular androgen binding protein content remained suppressed and serum androgen binding protein was elevated, indicating a prolonged defect in Sertoli cell function. These lesions were prevented by GnRH-A pretreatment. The present study demonstrates that GnRH-A pretreatment and subsequent TC implantation resulted in restoration of complete spermatogenesis in adult male rats given a 400-mg/kg cumulative dose of PCB. It is postulated that GnRH-A may ameliorate PCB induced Sertoli cell dysfunction and/or stimulate the number of spermatogonia to provide more proliferating cells ready for repopulation of the germinal epithelium following PCB injury. The differentiation of these spermatogonia was further supported by exogenous testosterone through certain unknown local mechanisms, resulting in the completion of spermatogenesis.

Hormonal treatment after cytotoxic therapy stimulates recovery of spermatogenesis.

Previous studies have shown that treatment of rats with gonadotropin-releasing hormone (GnRH) analogues or steroids either before exposure to procarbazine or radiation or after irradiation enhances subsequent levels of spermatogenesis. We demonstrate here that giving a GnRH agonist after procarbazine injection also enhances spermatogenesis and fertility. We also demonstrate that GnRH agonist stimulated recovery of spermatogenesis and fertility not only when the hormone was administered immediately after irradiation, but also at 20 weeks after irradiation, after the decline in spermatogenesis had occurred. These results suggest that GnRH agonist treatment given to azoospermic men after cytotoxic therapy for cancer may stimulate the recovery of spermatogenesis and fertility.

Cytotoxicity and DNA damage caused by the azoxy metabolites of procarbazine in L1210 tumor cells.

Procarbazine, a chemotherapeutic hydrazine, is thought to be metabolized to an alkylating species similar to methyl carbonium ion by multistep reactions involving cytochrome P-450, monoamine oxidase, and cytosolic enzymes. The DNA-damaging and cytotoxic potential of procarbazine and its metabolites in murine L1210 leukemia tumor cells in vitro was determined using alkaline elution techniques and extrapolation of growth curves. Neither procarbazine nor any of the chemical degradation products (except for the aldehyde derivative at high concentrations) caused significant amounts of DNA strand breakage. The primary enzymatic oxidation product, azo-procarbazine, did not produce strand breakage. However, exposure of the cells to either of the two isomers of azoxy-procarbazine led to significant DNA damage and cytotoxicity. DNA damage included both single-strand breaks and alkali-labile sites. At equimolar concentrations, the azoxy 2 isomer of procarbazine caused 14 to 20 times more DNA damage than did the azoxy 1 metabolite. When cell growth is expressed as percentage survival of L1210 cells, the azoxy 2 isomer was approximately 7-fold more toxic than the azoxy 1 metabolite. The other metabolites tested showed little or no cytotoxicity. L1210 cells were shown to contain little or no cytochrome P-450 or monoamine oxidase activity, which may account for the lack of toxicity of the parent drug or the primary oxidative metabolite, azo-PCZ, to these cells. The conversion of procarbazine to the azoxy-procarbazine isomers in vivo must occur in cells which contain these enzymes, such as liver. However, the azoxy isomers of procarbazine were metabolized in L1210 cells, presumably leading to the DNA or cytotoxic damage observed.