Mutation Research, 55 ( 1 9 7 8 ) 1--14 © E l s e v i e r / N o r t h - H o l l a n d B i o m e d i c a l Press

MUTAGENICITY, CARCINOGENICITY AND TERATOGENICITY OF PROCARBAZINE

I.P. L E E and R.L. D I X O N

National Institute o f Environmental Health Sciences, Research Triangle Park, N.C. 27709 U.S.A. (Received 15 D e c e m b e r 1977) (Revision received 12 May 1978) ( A c c e p t e d 22 May 1978)

Contents Introduction ................................................... Mutagenic e f f e c t s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Antifertility effects ............................................... Carcinogenic e f f e c t s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Teratogenic effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . M e c h a n i s m o f M I H - i n d u c e d m u t a g e n i c i t y , c a r c i n o g e n i c i t y and t e r a t o g e n i c i t y . . . . . . . . . Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements ............................................... References ....................................................

1 2 5 5 7 7 9 10 10

Introduction Although hydrazine (NH2NH2) and hydrazide compounds are generally recognized as toxic chemicals, causing liver damage, hypoglycemia, blood dyscrasias, and convulsions, they have proven extremely useful in the fields of aerospace and health. Hydrazine and its methyl derivatives are widely used as rocket fuels; substituted hydrazines and hydrazides have been synthesized in search of monoamine oxidase inhibitors which stimulate the central nervous system and elevate mood [99]. Since hydrazine and hydrazide compounds have a variety of pharmacological actions, hydrazine derivatives were tested for effectiveness against tumors, and some were found to have potent antitumor activity. Systematic variation of the functional group (R) in the general chemical formula CH3NHNHCH2R revealed Abbreviations: MIH, N-isopropyl-~-(2-methylhydrazino)-p-toluamide~ MOMP, nitrogen mustard, vincristine, methotrexate and prednisone; MOPP, nitrogen mustard, vincristine, MIH and prednisone.

-N-CCH2.H-NH-CH o

CH 3 PROCARBAZINE (MIH) Fig. 1. T h e s t r u c t u r e o f p r o c a r b a z i n e (MIH).

that the most active derivate synthesized is N-isopropyl~-(2-methylhydrazino)p-toluamide (MIH, Matulane ®, Natulan ®, R04-6467, NSC-77213); its structure is shown in Fig. 1. MIH was approved for clinical trials in 1969 and found effective alone or in multi-drug combination chemotherapy against Hodgkin's disease as well as other types of lymphomas, polycythemic vera, malignant melanoma, bronchogenic carcinoma, and multiple myeloma [87]. These therapeutic effects are accompanied by significant toxicity. In mice, rats and rabbits, the oral LDs0 of MIH is reported to be 1320 ± 66, 785 _+34, and 145 +_11.5 mg/kg respectively [74]. The major toxic effects of MIH are reversible hematopoietic supression [90] and hemolytic anemia [61]. In rodents MIH depletes plasma pyridoxal phosphate [64]; however, exogenous pyridoxine supplementation clinically has not been particularly effective in reversing various forms of MIH toxicity [18]. MIH neurotoxicity may be manifested by somnolence, depression and phychosis. Furthermore, MIH treatment potentiates the effects of barbiturates in mice, possibly depressing the central system directly and/or through the inhibition of hepatic microsomal mixed function oxidase(s) [51]. Moreover, antihypertensive and sympathomimetic effects may be potentiated in patients taking MIH and foods with high tyramine content [24]. Antispermatogenic [35,49] and antifertility effects of MIH have been demonstrated in male and female rodents [49,58] and reported clinically [83,92]. Although the evidence is not entirely conclusive, experimental data suggest that MIH toxicity includes chromosomal aberrations, which may account in part for its mutagenic effects. As for carcinogenic effects, some evidence correlates point mutation and carcinogenicity, but as yet no convincing evidence demonstrates that chromosomal aberrations lead to either carcinogenicity or teratogenicity. This review focuses upon the mutagenic, carcinogenic and teratogenic effects. Previously, several reviews on MIH have been reported [19,37,65,69, 72,78,87,89]. Mutagenic effects As early as in 1963, Rutishauser and Bollag [76] observed chromosomal aberration in Ehrlich ascites cells exposed to the procarbazine derivative, 1-methyl-2-benzylhydrazine phosphate. In further cytogenetic studies with this agent, chromatid translocations were observed in 3 types of mouse ascites tumors in vivo: Ehrlich, 2BF and L-1210 lymphocytic leukemic cells. Ehrlich tumor cell showed the higher frequency of translocation [91]. In vivo chromosomal breaks of the G2 type occurred in all 3 cell types although cells

from the same tumor differed greatly in their response to MIH. In contrast, in vitro exposure of these cells failed to induce chromosomal translocation, suggesting a requirement for metabolic activation [76,91]. MIH also failed to produce chromosomal translocation in in vitro human leukocytes. Spermatogenic cell death was reported after treatment of mice and rats with MIH; 6 days after treatment, leptotene, zygotene, and pachytene spermatocytes were dying with chromosomal aber~'ations [35]. Furthermore, the frequencies of chromatid aberrations in spermatogonia of male mice treated with MIH (200 and 800 mg/kg) were observed to increase with increasing dose of MIH [12]. However, non-disjunction in germ cells has not been observed thus far. In most cases, human chromosomal damage by procarbazine has been observed in .the lymphocytes and bone-marrow cells of patients treated for various diseases such as rheumatoid arthritis [93], acute myelofibrosis, peyronie [68], and various tumors [80]. MIH treatment of patients with acute myelofibrosis induced significant chromosomal abnormalities within blood cells. The structural defects included chromatid breaks, translocated D-chromosomes and dicentric chromosomes [68]. The chromosomes of lymphocyte cultures from the peripheral blood of rheumatoid arthritic patients receiving immunosuppressive therapy with procarbazine demonstrated chromatid breaks, gaps, isogaps, fragmentations, and dicentric chromosomes [93 ]. Approximately 60% of the lymphocyte cultures from patients receiving procarbazine had greater than 1% chromosomal aberration and 4% chromatid breaks and exchanges [80]. However, the percent of chromosomal aberration varied significantly even though the lymphocyte cultures were derived from patients receiving high doses of cytostatic agents with known chromosome-breaking activity. This inconsistency suggests that cytogenetic examination of lymphocyte cultures from exposed patients is not a reliable screening test for weak mutagens [80]. However, the conclusion of these studies may have been entirely different if the examination of chromosomal aberrations were carried out at an earlier time of lymphocyte culture (48-h instead of 72-h cultures) [80]. Other factors, intrinsic to combination chemotherapy for cancer, complicate the assessment of chromosomal aberrations in cells exposed to MIH. For example, children undergoing combination chemotherapy {including MIH) for cancer show a significant increase in nuclear aberrations in their oral epithelial cells [88]; patients receiving MIH for acute myelofibrosis have a high incidence of chromosomal aberration, such as breaks, translocation and dicentrics in metaphase [29,41,68]. However, most of these studies were performed using preparations of chromosomes from patients receiving more than one chemotherapeutic agent (often with radiation), thus introducing many variables which make it difficult to implicate a single agent in the production of chromosomal aberrations. Further complications arise from the variety of dose schedules utilized clinically and the differences in the interval between administration of MIH with other agents and examination of chromosomes; this interval varies from a few hours to weeks and even months. Nevertheless, it is clear that MIH can induce chromosomal damage in human cells. Mutagenicity studies with experimental animals have also demonstrated that

MIH induces dominant lethality in mice [27,30], as well as specific-locus mutations in spermatogonia [28]. Dominant lethality was chiefly associated with spermatids and spermatocytes, b u t MIH also inhibited the development of spermatocytes and spermatogonia. With a 400 mg/kg dose of MIH to mice, the induction of dominant-lethal mutations in spermatids was due mainly to post-implantation loss. However, doubling the dose reversed this effect, dramatically increasing preimplantation loss while the post-implantation loss was significantly reduced. Dominant-lethal mutation in spermatocytes was also associated with severe post-implantation loss. Again, doubling the dose reversed the effect. However, in male CD~F~mice treated with MIH (400 mg/ kg), dominant lethality was associated n o t only with spermatids (Golgi and acrosome phase of spermiogenesis) and all stages of spermatocytes b u t also with the t y p e B and intermediate spermatogonia [49]. This differential sensitivity to MIH may be attributable to mice strain differences. Several different types of genetic damage were observed in Drosophila melanogaster [12]. (1) sex-linked recessive lethals; (2) dominant lethals; (3) total and partial sex-chromosome loss; and (4) translocations. MIH is highly mutagenic in causing recessive-lethal mutations in all spermatogenic stages. With sperm, a clear-cut dose--response relationship was n o t apparent. However, with spermatids such a relationship was obtained. The induction of point mutation without any chromosomal aberrations was observed at low doses (0.5--10 mM) of MIH exposure [12] while at higher concentrations, the induction of recessive lethality was n o t a function of dose. A low induction of total sex-chromosome loss (XY) and dominant lethals was observed in metabolically active spermatids, b u t MIH failed to produce welldefined breakage events such as partial sex-chromosome loss (yL, y s ) and II-III translocations. The failure of MIH to induce chromosomal aberrations in Drosophila melanogaster m a y be due to a different degree of metabolic activation in insects than in mammals. The mutagenic effects of MIH appear to have both mutational and chromosomal effects which are dependent on MIH concentrations. The MIH concentrations required to increase the frequency of point mutations are far lower than the concentration required for the induction of chromosomal aberrations. Other mutagenic studies with MIH have shown variable results: MIH is mutagenic to mice as demonstrated b y dominant-lethal tests [27,30,49], b y the specific locus test [28], and b y the micro-nucleus assay [34]. Its mutagenicity in Drosophila melanogaster is indicated b y point mutations, sex-linked recessive lethals, dominant lethals, sex-chromosome loss, and translocations [12]. The marked differences in the mutational effects of MIH in the mammalian system versus the insect system may be attributed to differences in the metabolic activation as well as transport and permeability of active metabolite to germ cells of both organisms. However, MIH is one of the few mutagens which does not produce a positive response in the Ames test system [56,57]. This negative response may be attributed to the complex biotransformation of MIH, the in vitro hepatic microsomal enzyme activation needs to be compared to that observed in vivo.

Antifertility effects Bollag and Theiss [15] reported that all methylhydrazines tested in vivo caused marked depression o f spermatogenesis and led to testicular atrophy. Furthermore, 1,2-bis-(2-methylhydrazinomethyl)-benzene dihydrochloride, a methylhydrazine derivative which is devoid of any effect on hematopoiesis, caused spermatogenic cell death. Hilscher and Reichelt [35] also observed spermatocyte death in mice and rats after MIH treatment. However, no details were reported with regard to its effects on spermatogenesis. Studies with male mice by Lee and Dixon [48], based on both biochemical and serial mating techniques, demonstrated that a maximally effective single dose of MIH affected all spermatogenic cell types except late spermatids and mature spermatozoa, and significantly decreased fertility throughout a 55-day period. The delayed recovery of spermatogenesis was apparently due to an effect on non-differentiating stem cells (spermatogonia A) and/or an effect on spermatogonia type B cells. However, the possibility can not be excluded that the delayed recovery might be partly attributed to MIH's effect on other endocrine systems essential for the maintenance or normal spermatogenesis. In vitro inhibition of DNA and RNA synthesis required a much higher concentration of MIH than its metabolite, N-isopropyl-a-(2-methylazo)-ptoluamide [50]. In rat spermatogonial cells, the extent of DNA strand breaks and the DNA-repair time is MIH-concentration dependent [52]. Moreover, compared to alkylating agents, MIH-induced DNA damage takes a much longer time to repair. In vitro incubation of mouse spermatogonial cells with MIH (0.5--20 pmoles/ml) failed to result in unscheduled DNA synthesis while DNA repair synthesis was observed following in vivo treatment (100--400 mg/kg) [54]. These contrasting data suggest that procarbazine requires in vivo activation to have antispermatogenic effects. In human clinical studies, persistent azoospermia and antifertility effects associated with MIH administration in combination with other chemotherapeutic agents for treating Hodgkin's disease have been reported [92]. Sherins and DeVita [83] examined the fertility of 16 male lymphoma patients treated with various forms of intensive chemotherapy consisting of cytoxan, a combination of cytoxan, vincristine and prednisone, and MOPP {nitrogen mustard, vincristine, MIH and prednisone) or MOMP {nitrogen mustard, vincristine, methotrexate and prednisone) combination regimens. 2 Months to 4 years after the last course of chemotherapy, 10 of the 16 patients showed germinal aplasia with testicular biopsy revealing the presence of only Sertoli cells in the seminiferous tubules. In 2 patients, partial depletion of the germinal epithelium was found. Biopsy of one showed scattered spermatogonia, spermatocytes, and spermatids while the other had only 30% of tubules intact. Since these patients received multi-combination chemotherapy, it is difficult to determine to what extent MIH alone was involved in the male germ-cell damage. Carcinogenic effects MIH has been shown to be a potent, multiple carcinogen in rodents, inducing varieties of malignant tumors. In 1964, Kelly et al. [38] reported that MIH induced a high incidence of

leukemia and many other neoplasms in mice and mammary adenocarcinomas in Osborne--Mendel rats. Further studies [39] showed that a single and repeated administration of MIH by oral or intraperitoneal route induced tumors in Osborne--Mendel and Fischer rats. In the Osborne--Mendel strain, 90--100% of females and 70% of males developed mammary adenocarcinomas after a median latent period of 13--19 and 27 weeks respectively, 40% of male rats developed hemangioendotheliomas and hemangiomas of the spleen, and several rats developed pulmonary tumors. Carcinoma of the duodenum and jejenum, hemangioendotheliomas of the kidney, uterus and liver, leukemia, fibrosarcoma of the oral submucosa, and subcutaneous rhabdomyosarcoma were also reported. In Fischer rats, 20--60% of the females but none of the males developed mammary tumors with a median latent period of 34--43 weeks, 40% of the females and 30% of the males developed tumors of the kidney, 10% of the females developed tumors of the uterus, 15% of the females and 30% of the males developed squamous cell carcinoma of the ceruminous and sebaceous glands, and a few animals developed pulmonary tumors. Leiomyosarcoma of the jejenum, ganglioneuroma of the adrenal gland, lymphosarcoma, reticulum cell sarcoma of lymph nodes, retroperitoneal rhabdomyosarcoma, and a subcutaneous fibrosarcoma were also reported [39,40,63]. Deckers et al. [23] observed tumors of uterus, mammary gland and ear duct in R strain rats after MIH treatment. Other laboratory animals are also susceptible to malignant tumors when treated with MIH. Studies of comparative carcinogenicity of MIH, its metabolic products, hydrazines and isonicotinic acid hydrazide suggest that multiple pulmonary tumors in mice are induced by MIH metabolic products, particularly the azo and hydrazo derivative; leukemia was associated with MIH and azo derivative [40]. Among primates, MIH was associated with the development of myelogeneous leukemia in 2 of 26 monkeys treated from birth [1]; liver tumors developed in Rhesus and cynomolgus monkeys [2]. Indirect evidence, the increasing number of reports of a second tumor type arising in cancer patients during or after MIH chemotherapy, suggests that MIH may be carcinogenic in humans. A number of surveys of patients treated for Hodgkin's disease, multiple myeloma, or who underwent prolonged immunosuppressive therapy for nonmalignant conditions (e.g. kidney transplantation, chronic renal disease, systemic lupus erythematosus, psoriasis, and rheumatoid arthritis) indicate that drug-induced secondary neoplasms were, without exception, acute myeloid leukemia [3] and lymphomas [55,60, 85,81]. Hoover and Fraumeni [36] have recently pointed out that after prolonged immunosuppressive therapy for kidney transplantation, recipients face a 30-fold higher risk of developing lymphoma than their control population. Radiation therapy combined with MIH has been associated with several apparent instances of a second nonlymphoid malignancy including acute myelocytic leukemia in conjunction with Hodgkin's disease [5,16,77]. This effect is not, however, observed in all studies [75]. Recently in mice, potentiation of the carcinogenic action of MIH by ionizing radiation has been reported [4].

To date there is insufficient information regarding the carcinogenic potential of anti-tumor or immunosuppressive agents in humans. However, evidence suggests that MIH is carcinogenic. Nevertheless a relatively large number of cancer patients have received and benefited from these chemotherapeutic agents although a second primary malignancy developed in some patients during or after chemotherapy [84]. Teratogenic effects MIH has been shown to be teratogenic in the rat [21]. After a single intraperitoneal administration of MIH at doses ranging from 5--550 mg/kg to pregnant rats on days 5, 12, 14, and 17 of gestation, malformation such as tail and appendicular defects, cleft palate and shortened jaws were observed in the offspring. Doses of 25, 50 and 75 mg/kg were lethal and teratogenic to some offspring after a single treatment on day 5, 6, 9, or 12, but produced no effect on day 14 or 17 of gestation. Although MIH has been shown to be teratogenic in experimental animals, teratogenic effects of MIH in man are still uncertain. However, a case of human fetal renal malformation following the administration of nitrogen mustard, vincristine, MIH, and prednisone in early pregnancy for treatment of Hodgkin's disease has been reported [59], although another young Hodgkin's disease patient who received MIH during the first 38 days of gestation gave birth to a term infant which was apparently normal except for some hemangiomas on the extremities [981. There is also report that 2 of 3 pregnant women given the drug delivered normal infants; the third infant was delivered prematurely and succumbed as a result of respiratory arrest b u t was without apparent physical or clinical abnormality at birth [22,23]. Human teratogenic potential of antineoplastic drugs has been reported previously [62,82,86]. Mechanism of MIH-induced mutagenicity, carcinogenicity and teratogenicity To date, the molecular mechanism by which MIH and/or its metabolites produce their biological effect at the molecular level has n o t been completely elucidated. An earlier cytological investigation suggested that MIH-induced chromatic breaks in Erhlich ascites cells accompanied by prolonged mitotic cycles [76] were attributable to the in vivo formation of hydrogen peroxide from MIH in the presence of oxygen. MIH-induced DNA fragmentation did n o t occur in the presence of either nitrogen or catalase, again suggesting that hydrogen peroxide is responsible for DNA fragmentation w i t h o u t substantial destruction of the double helix [10,11]. In vitro incubation of mouse spermatogenic cells with varying concentrations of MIH inhibited equally the incorporation of [3H]thymidine and [3H]uridine, b u t L-leucine incorporation was not inhibited [49]. In contrast, freshly prepared MIH does n o t have any significant effect on DNA, RNA, or protein synthesis in Ehrlich ascites cells in vitro [31]. Evidence obtained from the in vitro time course studies of DNA and RNA synthesis suggests that the inhibitory c o m p o n e n t of nucleic acid synthesis was n o t due to hydrogen peroxide b u t rather to one or more substances formed during in vitro incubation of MIH at 37°C. Furthermore, the inhibitory action

of MIH and DNA synthesis was not antagonized by nucleosides, DNA, or RNA and was reversed by washing the cells free of MIH [31]. This finding was further confirmed by Gutterman et al. [33]. The in vivo effects of MIH were greater than its in vitro effect on the inhibition of DNA and RNA synthesis. DNA, RNA and protein synthesis are inhibited by MIH in Ehrlich ascites cells [31,33,42,76,94], in L5178Y cells [79], and in mouse spermatogenic cells [48,49]. In L5178Y cells, MIH inhibited thymidine incorporation into DNA maximally (70%) 1--3 h after MIH. By 24 h after the MIH, thymidine uptake into DNA returned to normal. However, in mouse spermatogenic cells, thymidine, uridine and LAeucine uptake into the spermatogenic cells were significantly inhibited by MIH. On days 5 through 15 following treatment, the maximum inhibition of uptake of all three substrates occurred. Maximal inhibition of thymidine uptake was 92% and rebounded to 120% of control 25 days after a single MIH treatment (400 mg/kg i.p.} and correlated well with male infertility [49]. Significant differences in the incorporation of thymidine and uridine into DNA and RNA suggest that spermatogenic cells are more sensitive to MIH when compared to other cell types. Inhibition of DNA synthesis could not be attributed to the inhibition of thymidine monophosphate kinase, or DNA polymerase [79]. Weitzel et al. [94] showed that relatively high concentrations of hydrogen peroxide and formaldehyde, both catabolic products of MIH, inhibited both DNA and RNA polymerase; the postulated metabolite of MIH, N-hydroxylated derivatives and formylhydrazine, also inhibited DNA and RNA polymerase [95,96]. MIH administered to rodents is metabolized via oxidative degradation through N-isopropyl-(2-methylazo)-p-toluamide [13,14], N-isopropyl(2-methylhydrazone)-p-toluamide [20], N-isopropyl-p-formylbenzamide and N-isopropylterethalmic acid [13,14]. The conversion of MIH to azoprocarbazine is thought to occur also in man and dog [66,67,71] and a similar reaction is reported to occur in vitro with liver microsomes [8,70]. In rat, the N-methyl group of MIH is rapidly converted to methane as well as formaldehyde and CO2 [7,8,9,25,26,70,73,96]. Weitzel et al. [96,97] postulated that metabolic intermediates, azomethines and N-hydroxymethyl derivatives, may alkylate cellular DNA, RNA and other macromolecules. Ful~her experimental studies showed that methyl-[14C]MIH methylated RNA and DNA nucleotides at various positions such as NT-methylguanine, 1-methylguanine, 5-methylcytosine, 1-methyladenine and thymine [17,43-47]. These metabolic pathways are presented in Fig. 2. The interaction of MIH with E. coli and BHK 21 DNA caused changes in the sedimentation of the DNA in alkaline gradient centrifugation. The sedimentation coefficient of the DNA decreased from 57 to 10S which corresponds to one chain break every 1200 nucleotides [6]. This degree of methylation of nucleotides is very small; mutagenic and carcinogenic consequences remain to be determined. However, it has recently been shown that in vitro exposure of rat spermatogenic cells and fibroblasts [ 53] to MIH {100, 200 and 400 mg/kg) caused extensive single strand DNA breaks; the duration of repair was dose dependent. At the lower dose of MIH, DNA repair in germ cells is prolonged over 5 days [52]. It is possible that MIH-induced mutagenic, carcinogenic,

CH3

[C

Procorbozine(MIH)

H202

CH3

N-isopropyl-o.-(2 - methylozo)- p- toluamide isomerizofion

(H'*,OH-)

H C--NH-C ' ~

CH3

~,I~•~I-I~-~,~n,

N-isopropyl-o.-(2-methylhydrozone)-_p-toluamide .,,~- H20 C02J)[

NH2NH2-J 1 CH3NHNHz N-isopropyl- p- formylbenzomide

methylhydrazine

aldehyde oxidase

H202

CH~ +NH2 +H" CH4 + N~, methane

CH3

N-isopropylterethalrnicacid

Fig. 2. T h e m e t a b o l i c p a t h w a y of p r o c a r b a z i n e (MIH).

and teratogenic effects might be attributed to DNA damage as a consequence of DNA alkylation and subsequent DNA strand breaks or inter-, intraocrosslinking between the DNA strands and/or between DNA and histone and/or non-histone proteins. Further studies are needed to elucidate the underlying mechanisms of MIH-induced point mutation, chromosomal breaks, as well as its mutagenicity, carcinogenicity and teratogenicity.

Concluding remarks This paper reviews the deleterious effects of MIH on both experimental animals and man. Iatrogenic side effects of MIH in the treatment of various neoplastic and non-neoplastic diseases with respect to mutagenic effects, antifertility effects, carcinogenic and teratogenic effects are presented. Both experimental and clinical studies strongly suggest that MIH is capable of inducing point-mutation chromosomal damage, mutagenicity, antifertility effects, carcinogenicity and teratogenicity. The molecular mechanism by which MIH might produce these biological effects at the molecular level has not been

10 completely elucidated. But it has been experimentally shown that after exposure to MIH and/or itg metabolites, DNA and R N A are alkylated at various positions of the nucleotides. This process has been postulated to be a possible mechanism of mutagenic, carcinogenic and teratogenic action; however, the precise mode and/or modes of MIH action with respect to point mutation, chromosomal breakage, mutagenicity, carcinogenicity and teratogenicity remain to be further elucidated. Although clinical consequences of MIH-induced chromosomal damage are n o t understood at present, most clinicians are willing to accept the mutagenic risk. However, it is important for clinicians to stress the need for genetic counseling for all individuals who are going to be or have been exposed to chemotherapeutic agents possessing mutagenic potential. Antifertility effects of MIH can be either reversible or irreversible, depending upon both the dose and duration of chemotherapy, especially in treating Hodgkin's disease. Nevertheless, when the issue is the prolongation of life, the risk of antifertility as a possible consequence is usually acceptable. Fortunately, risks from teratogenic effects of MIH in the human are very small and can easily be circumvented. Nevertheless, the effects must also be considered by clinicians. Carcinogenic effects of MIH have been shown in experimental animals; in regard to man, our knowledge is uncertain. However, indirect evidence, the reports of secondary tumors arising in cancer patients during or after chemotherapy, is becoming available. Thus, the potential carcinogenicity of MIH in man continues to be the most troublesome of its various side effects. Cancer patients receiving combination chemotherapy which includes MIH should be informed of the possible genetic and carcinogenic risks to themselves as well as to their offspring. Acknowledgements The authors wish to thank Glenn Williams and Frederick Lee for their invaluable assistance in the preparation of the manuscript. References 1 Adamson, R.H., Carcinogenicity studies with procarbazine, in: S.K. Carter (Ed.), Proceedings of the Chemotherapy Conference on Procarbazine (Matniane: N S C 77213): Development and Application, U.S. Government Printing Office, Washington, D.C,, 1971, pp. 29--33. 2 Adamson, R.H., P. Correa, C.F. Smith, S.T. Yancey and E.W.Dalgard, Induction of tumors in m o n keys by chemical carcinogens-correlationof serum alpha-fetoprotein and appearance of livertumors, Proc. A m . Assoc. Cancer Res., 14 (1973) 42. 3 Andersen, E., and A. Videbaek, Stem cell leukemia in myelomatosis, Scand. J. Haematol., 7 (1970) 201--207. 4 Arseneau, J.C., E. Fowler and R.F. Bakemier, Synergistic carcinogenic effect of procarbazine and ionizing radiation in C D 2 F 1 mice, Proc. A m . Assoc. Cancer Res., 16 (1975) 120. 5 Arseneau, J.C., R.W. Sponzo and D.L. Levin, N o n l y m p h o m a t o u s malignant tumors complicating Hodgkin's disease. Possible associationswith intensive therapy, N e w Engl. J. Med., 287 (1972) 1119-1122. 6 Audubert, F., Action du 1-methyl-a(p-isopropyl-carbarnoyl-benzyl)-hydrazine sur le D N A in vitro et in vivo (cenules en culture),Biochim. Biophys. Acta, 281 (1972) 507--513. 7 Baggiolini, M., M.H. Bickel and R.S. Messiha, Demethylation in vivo of Natulan, a tumor-inhibiting methylhydrazine derivative,Zxperientia, 21 (1965) 334--336.

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