Fd Chem. Toxic. Vol. 24, No. 6/7, pp. 705--706, 1986 0278-6915/86 $3.00+0.00 Printed in Great Britain. All rights reserved Copyright © 1986 Pergamon Journals Ltd

GENOTOXICITY OF PROCARBAZINE IN MAMMALIAN CELLS IN VITRO

W. Su~R Sandoz Ltd, Pharmaceutical Department, Toxicology, CH-4002 Basel, Switzerland

Introduction

Procarbazine (Natulan ®, Hoffman-La Roche Ltd), a well-known cytostatic, is primarily used in the treat- ment of Hodgkin's disease. Like many cytostatic agents, procarbazine has been found to be carcino- genic, teratogenic and mutagenic in various species. Its mutagenicity has been studied extensively and it has been found to be a strong mutagen in all mam- malian in vivo tests, but many false-negative results in in vitro assays have been reported. Most in vitro work was performed using bacteria or yeast as the test organism (Hansmann, 1982). However only the use of hepatocytes as a system for metabolic activation allowed the mutagenicity of procarbazine to be de- tected at relatively low concentrations (Malaveille, Brun & Bartsch, 1983). Procarbazine therefore ap- peared to be a good model compound for in- vestigating the use of hepatocytes as a system for metabolic activation and as target cells for measuring DNA repair induction (unscheduled DNA synthesis--UDS).

HGPRT assay

The hypoxanthine-guanine phosphoribosyl trans- ferase assay in V79 Chinese hamster fibroblasts (the V79 HGPRT assay) was used to measure the in- duction of gene mutations. Procarbazine had a weak mutagenic effect in the cytotoxic dose range (beyond 60001zg/ml with a treatment time of 3 hours) in the absence of a metabolic activation system and in the presence of S-9 mix prepared from non-induced rat liver. When hepatocytes prepared from non-induced rats were used for metabolic activation, increased mutant frequencies were observed with concen- trations as low as 2/~g/ml, i.e. I000 times lower than the lowest cytotoxic concentration, which was 2000/~g/ml under these conditions. The maximal effect was observed at 200/~g/ml, with 53.8 mutant clones/106 cells compared to a figure of 4.4 in the control group.

The use of liver preparations from Aroclor 1254-pretreated animals led to a large increase in the mutagenic effect irrespective of whether S-9 homoge- nate or hepatocytes were used. Aroclor-induced S-9 mix led to mutagenic effects quantitatively similar to the effects observed using untreated hepatocytes. Using hepatocytes from Aroclor 1254-pretreated rats, an increase from the control level of 6.6 mutants/106 cells to 358 mutants/106 cells was found at 600pg procarbazJne/ml. This effect was seven times greater than that in the presence of uninduced

hepatocytes. In contrast to the situation in the pres- ence of S-9 mix, where an increase in mutagenicity was observed in the cytotoxic dose range, the mu- tagenic effect in the presence of hepatocytes dropped considerably below the maximum if the test- compound concentrations reached the cytotoxic dose range. This can be explained by assuming that cyto- toxicity interfered with proper metabolic activation.

Induction of DNA repair

UDS measurement in rat hepatocyte primary cul- tures was performed in parallel both by auto- radiography (Williams, Laspia & Dunkel, 1982) and by liquid scintillation counting after isolation of nuclei (Althaus, Lawrence, Sattler & Longfellow, 1982), using the same batch of cells. Toxic effects (i.e. pycnotic nuclei) were observed at 6000/~g/ml using non-induced hepatocytes and at 1250#g/ml with hepatocytes derived from Aroclor 1254-pretreated animals. A drop in [~H] thymidine incorporation was taken as an indication of cytotoxicity in mea- surements using scintillation counting. This effect occurred, irrespective of whether or not the cells were pretreated, between 2000 and 6000/~g/ml. Pro- carbazine was found to induce DNA repair in non- treated hepatocytes at 600 l~g/mi (one of two experi- ments) and at 2000/~g/ml if analysis was by autoradiography.

An increase in [3H]thymidine incorporation ob- served in one of two scintillation experiments at 2000/zg/ml was statistically significant by Student's t test.

Hepatocytes were prepared 1 and 5 days after Aroclor 1254 treatment. Both batches of ceils allowed the detection of procarbazine-induced DNA repair, at concentrations of 37 and 39/~ g/ml respectively, by either of the evaluation techniques.

Condusions

Procarbazine was found to be mutagenic in the V79 HGPRT assay and induced DNA repair in rat hepatocyte primary cultures. Hepatocytes were far more efficient than S-9 liver homogenate in producing mutagenic procarbazine metabolites. The use of Aro- clot 1254-pretreated liver preparations led to a strong increase in the mutagenic and DNA repair-inducing effects of procarbazine. Depending on the type of experiment, cytotoxicity was either weakly affected or was not affected at all by the Aroclor 1254 pre- treatment.

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Hepatocytes prepared 1 day or 5 days after Aroclor 1254 pretreatment were equally sensitive indicators of procarbazine genotoxicity. However, higher yields and better attachment on the tissue-culture plates made hepatocytes prepared 1 day after Aroclor 1254 treatment a much better system for studying geno- toxicity in induced cells than hepatocytes prepared 5 days after pretreatment. UDS measurements by auto- radiography and by liquid scintillation counting led to the same conclusions, although in the case of non-induced cells the effect was more pronounced using the autoradiographic procedure.

REFERENCES

Althaus F. R., Lawrence S. D., Sattler G. L. & Longfellow

D. G. (1982). Chemical quantification of unscheduled DNA synthesis in hepatocytes as an assay for the rapid screening of chemical carcinogens. Cancer Res. 42, 3010.

Hansmann J. (1982). Comparative mutagenicity of pro- carbazine (Natulan~). In Comparative Chemical Mu- tagenicity. Edited by F. J. de Serres & M. D. Shelby. p. 977. Plenum Press, New York.

Malaveille C., Brun G. & Bartsch H. (1983). Studies on the efficiency of the Salmonella/rat hepatocyte assay for the detection of carcinogens as mutagens: activation of 1,2-dimethylhydrazine and procarbazine into bacterial mutagens. Carcinogenesis 4, 449.

Williams G. M., Laspia M. F. & Dunkel V. C. (1982). Reliability of the hepatocyte primary culture/DNA repair test in testing of coded carcinogens and noncarcinogens. Mutation Res. 97, 359.