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Mutagenicity and carcinogenicity of mono- and diacetyl hydrazine
235
Cancer Letters, 23 (1984) 235-240 Elsevier Scientific Publishers Ireland Ltd.
MUTAGENICITY AND CARCINOGENICITY DIACETYL HYDRAZINE
SUMATI V. BHIDE, ERAWATI B. BHALERAO, MARU
OF MONO- AND
ANITA V. SARODE and GIRISH B.
Carcinogenesis Division, Cancer Research Institute, Tata Memorial Centre, Parel, Bombay 4 00 0 12 (India) (Received 27 February 1984) (Revised version received 4 May 1984) (Accepted 7 May 1984)
SUMMARY
Two hydrazine derivatives, monoacetyl hydrazine (MAH) and diacetyl hydrazine (DAH), have been tested for mutagenic response in the Salmonellu/ mammalian microsome assay and micronucleus test. MAH but not DAH, increased the revertant mutants in TAlOO and TA1535 and also increased the frequency of micronuclei in polychromatic erythrocytes. Gavage administration of MAH but not of DAH, resulted in increased incidence of lung tumors. These observations record for the first time the mutagenicity/ carcinogenicity of MAH which is one of the metabolites of isoniazid in animals and humans.
INTRODUCTION
In the past years many hydrazines, hydrazides and hydrazones have been found to be carcinogenic in animals [15]. Therefore a role of hydrazine derivatives in the etiology of human cancer cannot be excluded. Synthetic substituted hydrazines are present in the environment and are used in industry and agriculture [ 71. Moreover, various hydrazines have been used or are currently in use as therapeutic drugs for the treatment of hypertension, depression, tuberculosis and cancer [6] and new drugs with a hydrazine moiety are frequently proposed by pharmaceutical industries [lO,ll]. The antituberculosis drug, isoniazid (INH), undergoes a major metabolic change in which it is acetylated to yield 1-acetyl-2-isonicotinoyl hydrazine (A-INH) which is the principal metabolite of INH in man, and it is further shown to be metabolized possibly through hydrazine into ammonia in some mammalian species [4,9]. Recently acetyl hydrazines have been positively identified in biological fluids of either laboratory animals or humans given 0304-3835/84/$03.00 0 1984 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland
236
isoniazid [ 8,13,14]. Acetyl hydrazine is hepatotoxic in animals [ 21 and is known to be eliminated from the body as free mono- or diacetyl hydrazine or as hydrazones of a-oxoglutaric acid and pyruvic acid. It may also be further metabolized by microsomal enzymes and excreted. In view of the possibility that, in addition to those already tested, other hydrazine derivatives might possess carcinogenic activity, the present study was performed to investigate 2 hydrazine derivatives, MAH and DAH, for their mutagenic and carcinogenic activity. Mutagenicity was tested in 2 short term tests, namely Ames test and micronucleus test. Inhibition of testicular DNA synthesis in Swiss mice was used as a supportive test to see if the test substances affected germ cells. All these effects have been qualitatively correlated with tumorigenic activity in Swiss mice. MATERIALS
AND METHODS
MAH (CH3CONHNH2, mol. wt. 74.08, LD5,, 150 mg/kg) and DAH (CH3CONHNHCOCH3, mol. wt. 116.12, LDsO 3000 mg/kg) were obtained from Ega Chemie, F.R.G. Inbred Swiss mice employed in the present investigations were from the animal colony of the Cancer Research Institute. Animals were kept on standard laboratory diet and water ad libitum. Mutagenic
activity
in the Ames test
Mutagenic activity was investigated with the Salmonetfalmicrosome test using water solutions of MAH and DAH at concentrations ranging from 0.5 to 5 mg/plate. Reversion of histidine auxotrophs of S. typhimurium strains TA1535, TAlOO, TA98 and TA92 were assayed in the absence and presence of S9 mixture [1] containing postmitochondrial (S9) fractions from animals treated i.p. with Arochlor 1254. The test chemical along with 0.1 ml of an overnight growth culture (usually adjusted to 2 X lo8 cells/O.1 ml of the inoculum) with or without S9 mixture, were transferred to molten agar at 45°C and poured over nutrient agar in petriplates containing traces of histidine and biotin. Number of bacteria in 0.1 ml of culture were determined by plating serially diluted culture in presence of histidine and biotin (0.1 M). The histidine revertants were counted after 48 h incubation at 37°C. Spontaneous revertants in each strain both in the presence and absence of S9 mixture were determined. Results are mean of 4 plates in each experiment. Micronucleus
test
In these experiments, 6-8-week-old Swiss male mice (20-30 g) were injected i.p. twice with 120 mg/kg MAH or 2400 mgfkg DAH at an interval of 24 h. Control mice received distilled water. Six hours after the second injection, animals were killed, bone marrow preparations were made, stained
237
in May-Grunwald Giemsa and mounted in Euparol as described [ 121. About 4000 polychromatic and normochromatic erythrocytes were scored per animal. Results are mean f S.E. of 4 animals in each group. Mouse testicular DNA synthesis inhibition test was performed as described by Friedman and Staub [ 51. A single i.p. injection of MAH (150 mg/kg) or DAH (3000 mg/kg) was given to Swiss male mice weighing 23-28 g. Results expressed as cpm/pg DNA are mean f S.E. of 4 observations in each group. Experimental mice were randomly distributed to various groups so as to have the same number of males and females in each group (total 30 or 40). Animals were fed with MAH or DAH (0.8 mg or 1.1 mg/day per mouse) or distilled water 5 times a week by stomach tube until they died or were killed. Continuous treatment with 1.1 mg/day per mouse of MAH or DAH was found to be toxic and only 10 animals survived. In another group of animals 1.1 mg/day per mouse of MAH or DAH was fed 5 times a week for 8 months only and animals were kept under observation. Lungs were examined macroscopically and fixed in 10% formalin. Tissues were processed by routine histological process and sections (6 pm) were stained with hematoxylin and eosin. Statistical analysis was performed by the x2-test. RESULTS
AND DISCUSSION
In the plate incorporation test MAH was mutagenic in the absence of S9 mixture for strains TAlOO and TA1535 with dose related effects (Fig. 1). Addition of S9 mixture decreased the number of induced revertants in both the strains but was still found to be positive. While in the case of DAH no increase of revertants over controls was detected up to 5 mg. The results indicate MAH to be a base pair substitution mutagen. Number of revertants decreased in the presence of S9 and this may possibly be due to conversion of MAH to DAH. The average values for the frequency of micronuclei in polychromatic erythrocytes are shown in Table 1. It is seen from the data that there is significant increase in the frequency of micronuclei in the polychromatic erythrocyte of the MAH treated group while a very marginal difference observed in the case of DAH was not statistically significant. The polychromatic to normochromatic ratio which gives the mitotic index was not significantly affected by the administration of MAH or DAH and it remained unaltered. Thus these results suggest that. under the experimental conditions described only MAH induced clastogenic and/or chromosomal damaging effect as revealed by the micronucleus test. Values expressed as cpm/r.lg DNA on the effect of MAH and DAH on the testicular DNA biosynthesis, i.e. 2.17 f 0.02 and 3.09 +_0.23 for MAH and DAH, respectively, versus 3.0 + 0.19 for the control, suggest that only MAH showed significant inhibitory effect on the incorporation of [ 3H] thymidine while DAH did not. A similar type of inhibition was also observed in lungs from MAH treated mice but not in DAH treated mice [3].
238
1600
1400
-
1600
-
1000
-
600
-
600
-
1 0’ a t! r 2 + a ? E
400
-
I
1
I
0
0.5
MAH
I
I
1.0
e.0
(m9)
Fig. 1. Dose response curves obtained with varying amounts of MAH in the presence (- - -) and absence ( -) of S9 mixture in S. typhimuriumstrains TAlOO (0) and TA1535 (x).
TABLE 1 MUTAGENICITY
OF METABOLITES
OF INH USING THE MICRONUCLEUS TEST
No.
Compound
Dose/ animal (mg)
% Polychromatic erythrocytes with micronucleus
P/N ratioa
1 2 3
Distilled water MAH DAH
0.2 2.4 48
0.25 f 0.05 0.68 f. 0.02* 0.36 10.03
1.0 1.09 1.1
Results are expressed as mean f S.E. of 4 animals in each group. a P/N ratio, ratio of polychromatic to normochromatic cells. *Statistically significant when compared with controls, P < 0.05.
239
These 2 compounds were further tested in a small group of animals for potential carcinogenic effects. Table 2 denotes the lung tumor incidence in various treatment groups. It is seen that in the group receiving 0.8 mg/day per mouse only MAH administration resulted in an increase in lung tumors from 13% to 50% while no tumors were observed in DAH treated groups. In the group receiving 1.1 mg/day per mouse continuously, only 10 animals survived up to the age of 16-22 months. The lung tumor incidence was 70% and 30%, respectively, in MAH and DAH treated groups, and in the group receiving 1.1 mg/day per mouse for only 8 months, the incidence was 71.1% and lo%, respectively. Since the number of animals in each group was small we have combined the 2 sexes for comparison between control and treated groups. This was done because the tumor incidence in treated male and female groups was comparable. For example in the 0.8 mg MAH treated group, the tumor incidence was 4/8 in males and 5/9 in females. The difference in the incidence of lung tumors between control and DAH is not statistically significant but difference between control and MAH group is significant. Tumor types observed were adenoma and papillary adenocarcinoma. Some of these tumors were transplantable. Few liver hemangiomas were observed in control as well as other groups and the difference was not statistically significant and hence not included in tumor incidence. The difference observed between mutagenicity and carcinogenicity of MAH and DAH does not appear to be due to difference in the amount used (different molecular weights) since both were used near or at LD5,, dose in short term tests while in long term studies they were toxic and hence it was difficult to use various doses. TABLE 2 TUMOR INCIDENCE IN SWISS MICE TREATED WITH MAH AND DAH Group
MAH Male
Distilled water control 0.8 mg/day per mouse continuous 1.1 mg/day per mouse continuous 1.1 mg/day per mouse for 8 months only
Female
Combined tumor incidence (%I
Male
Female
2118
3120
O/10
O/10
Combined tumor incidence (%I 5138 (13) o/21 (0)
2118
3120
418
519
7110
-
7110 (70)
3110
-
3110 (30)
6111
17124 (71)
2/11
018
2119 (10)
11/13
5138 (13) 9117 (51)
DAH
240
Thus the preliminary observations record for the first time the mutagenicity/carcinogenicity of MAH which is one of the metabolites of INH in animals and humans. REFERENCES 1 Ames, B.N., McCann, J. and Yamasaki, E. (1975) Methods for detecting carcinogens and mutagens with the Solmonellu/mammalian microsome mutagenicity test. Mutat. Res., 31, 347-364. 2 Balo, J. (1979) Role of hydrazine in carcinogenesis. In: Advances in Cancer Research, Vol. 30, pp. 151-164. Editors: G. Klein and S. Weinhouse. Academic Press, London. 3 Bhalerao, E.B. (1982) Studies on the possible mechanism of isoniazid carcinogenesis, Ph.D. thesis, University of Bombay. 4 El Masri, A.M., Smith, J.N. and Williams, R.T. (1958) Studies in detoxification 75. Further observations on the metabolism of hydrazines of aromatic acids. Biochem. J., 68,587-592. 5 Friedman, M.A. and Staub, J. (1976) Inhibition of mouse testicular DNA synthesis by mutagens and carcinogens as a potential simple mammalian assay for mutagenesis. Mutat. Res., 37,67-76. 6 Goodmann, L.S. and Gilman, A. (1975) The Pharmacological Basis of Therapeutics, 5th edn., MacMillan Publishing Co., New York. 7 IARC (1974) IARC monographs on the evaluation of carcinogenic risk of chemicals to man, Vol. 4, pp. 125-179. International Agency for Research on Cancer, Lyon. 8 Noda, A., Goromaru, T., Matsuyama, K., Sogabe, K., Hsu, K.Y. and Iguchi, S. (1978) Quantitative determination of hydrazines derived from isoniazid in patients. J. Pharm. Dyn., 1, 132-141. 9 Porcellati, G. and Preziosi, P. (1954) Transformazione biologica dell’idrazide dell’acido isonictinico. Enzymologia, 17, 47-54. 10 Salvadeo, A., Villa, G., Segagni, S. and Criscuolo, D. (1979) Long-term study of oxideralazine in hypertensive patients, Arzneim Forsch., 29, 1753-1755. 11 Schenker, E. and Salzman, R. (1979) Bicyclic 3-hydrazino pyridazines with antihypertensive action. Arzneim Forsch., 29, 1835-1843. 12 Schmid, W. (1975) The micronucleus test. Mutat. Res., 31, 9-15. 13 Timbrell, J.A., Wright, J.M. and Baillie, T.A. (1977) Monoacetyl hydrazine as a metabolite of isoniazid in man. Clin. Pharmacol. Ther., 22, 602-608. 14 Timbrell, J.A., Wright, J.M. and Smith, G.M. (1977) Determination of hydrazine metabolites of isoniazid in human urine by gas chromatography. J. Chromatogr., 138, 165-172. 15 Toth, B. (1980) Actual new cancer causing hydrazines, hydrazides and hydrazones. J. Cancer Res. Clin. Oncol., 97, 97-108.
Cancer Letters, 23 (1984) 235-240 Elsevier Scientific Publishers Ireland Ltd.
MUTAGENICITY AND CARCINOGENICITY DIACETYL HYDRAZINE
SUMATI V. BHIDE, ERAWATI B. BHALERAO, MARU
OF MONO- AND
ANITA V. SARODE and GIRISH B.
Carcinogenesis Division, Cancer Research Institute, Tata Memorial Centre, Parel, Bombay 4 00 0 12 (India) (Received 27 February 1984) (Revised version received 4 May 1984) (Accepted 7 May 1984)
SUMMARY
Two hydrazine derivatives, monoacetyl hydrazine (MAH) and diacetyl hydrazine (DAH), have been tested for mutagenic response in the Salmonellu/ mammalian microsome assay and micronucleus test. MAH but not DAH, increased the revertant mutants in TAlOO and TA1535 and also increased the frequency of micronuclei in polychromatic erythrocytes. Gavage administration of MAH but not of DAH, resulted in increased incidence of lung tumors. These observations record for the first time the mutagenicity/ carcinogenicity of MAH which is one of the metabolites of isoniazid in animals and humans.
INTRODUCTION
In the past years many hydrazines, hydrazides and hydrazones have been found to be carcinogenic in animals [15]. Therefore a role of hydrazine derivatives in the etiology of human cancer cannot be excluded. Synthetic substituted hydrazines are present in the environment and are used in industry and agriculture [ 71. Moreover, various hydrazines have been used or are currently in use as therapeutic drugs for the treatment of hypertension, depression, tuberculosis and cancer [6] and new drugs with a hydrazine moiety are frequently proposed by pharmaceutical industries [lO,ll]. The antituberculosis drug, isoniazid (INH), undergoes a major metabolic change in which it is acetylated to yield 1-acetyl-2-isonicotinoyl hydrazine (A-INH) which is the principal metabolite of INH in man, and it is further shown to be metabolized possibly through hydrazine into ammonia in some mammalian species [4,9]. Recently acetyl hydrazines have been positively identified in biological fluids of either laboratory animals or humans given 0304-3835/84/$03.00 0 1984 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland
236
isoniazid [ 8,13,14]. Acetyl hydrazine is hepatotoxic in animals [ 21 and is known to be eliminated from the body as free mono- or diacetyl hydrazine or as hydrazones of a-oxoglutaric acid and pyruvic acid. It may also be further metabolized by microsomal enzymes and excreted. In view of the possibility that, in addition to those already tested, other hydrazine derivatives might possess carcinogenic activity, the present study was performed to investigate 2 hydrazine derivatives, MAH and DAH, for their mutagenic and carcinogenic activity. Mutagenicity was tested in 2 short term tests, namely Ames test and micronucleus test. Inhibition of testicular DNA synthesis in Swiss mice was used as a supportive test to see if the test substances affected germ cells. All these effects have been qualitatively correlated with tumorigenic activity in Swiss mice. MATERIALS
AND METHODS
MAH (CH3CONHNH2, mol. wt. 74.08, LD5,, 150 mg/kg) and DAH (CH3CONHNHCOCH3, mol. wt. 116.12, LDsO 3000 mg/kg) were obtained from Ega Chemie, F.R.G. Inbred Swiss mice employed in the present investigations were from the animal colony of the Cancer Research Institute. Animals were kept on standard laboratory diet and water ad libitum. Mutagenic
activity
in the Ames test
Mutagenic activity was investigated with the Salmonetfalmicrosome test using water solutions of MAH and DAH at concentrations ranging from 0.5 to 5 mg/plate. Reversion of histidine auxotrophs of S. typhimurium strains TA1535, TAlOO, TA98 and TA92 were assayed in the absence and presence of S9 mixture [1] containing postmitochondrial (S9) fractions from animals treated i.p. with Arochlor 1254. The test chemical along with 0.1 ml of an overnight growth culture (usually adjusted to 2 X lo8 cells/O.1 ml of the inoculum) with or without S9 mixture, were transferred to molten agar at 45°C and poured over nutrient agar in petriplates containing traces of histidine and biotin. Number of bacteria in 0.1 ml of culture were determined by plating serially diluted culture in presence of histidine and biotin (0.1 M). The histidine revertants were counted after 48 h incubation at 37°C. Spontaneous revertants in each strain both in the presence and absence of S9 mixture were determined. Results are mean of 4 plates in each experiment. Micronucleus
test
In these experiments, 6-8-week-old Swiss male mice (20-30 g) were injected i.p. twice with 120 mg/kg MAH or 2400 mgfkg DAH at an interval of 24 h. Control mice received distilled water. Six hours after the second injection, animals were killed, bone marrow preparations were made, stained
237
in May-Grunwald Giemsa and mounted in Euparol as described [ 121. About 4000 polychromatic and normochromatic erythrocytes were scored per animal. Results are mean f S.E. of 4 animals in each group. Mouse testicular DNA synthesis inhibition test was performed as described by Friedman and Staub [ 51. A single i.p. injection of MAH (150 mg/kg) or DAH (3000 mg/kg) was given to Swiss male mice weighing 23-28 g. Results expressed as cpm/pg DNA are mean f S.E. of 4 observations in each group. Experimental mice were randomly distributed to various groups so as to have the same number of males and females in each group (total 30 or 40). Animals were fed with MAH or DAH (0.8 mg or 1.1 mg/day per mouse) or distilled water 5 times a week by stomach tube until they died or were killed. Continuous treatment with 1.1 mg/day per mouse of MAH or DAH was found to be toxic and only 10 animals survived. In another group of animals 1.1 mg/day per mouse of MAH or DAH was fed 5 times a week for 8 months only and animals were kept under observation. Lungs were examined macroscopically and fixed in 10% formalin. Tissues were processed by routine histological process and sections (6 pm) were stained with hematoxylin and eosin. Statistical analysis was performed by the x2-test. RESULTS
AND DISCUSSION
In the plate incorporation test MAH was mutagenic in the absence of S9 mixture for strains TAlOO and TA1535 with dose related effects (Fig. 1). Addition of S9 mixture decreased the number of induced revertants in both the strains but was still found to be positive. While in the case of DAH no increase of revertants over controls was detected up to 5 mg. The results indicate MAH to be a base pair substitution mutagen. Number of revertants decreased in the presence of S9 and this may possibly be due to conversion of MAH to DAH. The average values for the frequency of micronuclei in polychromatic erythrocytes are shown in Table 1. It is seen from the data that there is significant increase in the frequency of micronuclei in the polychromatic erythrocyte of the MAH treated group while a very marginal difference observed in the case of DAH was not statistically significant. The polychromatic to normochromatic ratio which gives the mitotic index was not significantly affected by the administration of MAH or DAH and it remained unaltered. Thus these results suggest that. under the experimental conditions described only MAH induced clastogenic and/or chromosomal damaging effect as revealed by the micronucleus test. Values expressed as cpm/r.lg DNA on the effect of MAH and DAH on the testicular DNA biosynthesis, i.e. 2.17 f 0.02 and 3.09 +_0.23 for MAH and DAH, respectively, versus 3.0 + 0.19 for the control, suggest that only MAH showed significant inhibitory effect on the incorporation of [ 3H] thymidine while DAH did not. A similar type of inhibition was also observed in lungs from MAH treated mice but not in DAH treated mice [3].
238
1600
1400
-
1600
-
1000
-
600
-
600
-
1 0’ a t! r 2 + a ? E
400
-
I
1
I
0
0.5
MAH
I
I
1.0
e.0
(m9)
Fig. 1. Dose response curves obtained with varying amounts of MAH in the presence (- - -) and absence ( -) of S9 mixture in S. typhimuriumstrains TAlOO (0) and TA1535 (x).
TABLE 1 MUTAGENICITY
OF METABOLITES
OF INH USING THE MICRONUCLEUS TEST
No.
Compound
Dose/ animal (mg)
% Polychromatic erythrocytes with micronucleus
P/N ratioa
1 2 3
Distilled water MAH DAH
0.2 2.4 48
0.25 f 0.05 0.68 f. 0.02* 0.36 10.03
1.0 1.09 1.1
Results are expressed as mean f S.E. of 4 animals in each group. a P/N ratio, ratio of polychromatic to normochromatic cells. *Statistically significant when compared with controls, P < 0.05.
239
These 2 compounds were further tested in a small group of animals for potential carcinogenic effects. Table 2 denotes the lung tumor incidence in various treatment groups. It is seen that in the group receiving 0.8 mg/day per mouse only MAH administration resulted in an increase in lung tumors from 13% to 50% while no tumors were observed in DAH treated groups. In the group receiving 1.1 mg/day per mouse continuously, only 10 animals survived up to the age of 16-22 months. The lung tumor incidence was 70% and 30%, respectively, in MAH and DAH treated groups, and in the group receiving 1.1 mg/day per mouse for only 8 months, the incidence was 71.1% and lo%, respectively. Since the number of animals in each group was small we have combined the 2 sexes for comparison between control and treated groups. This was done because the tumor incidence in treated male and female groups was comparable. For example in the 0.8 mg MAH treated group, the tumor incidence was 4/8 in males and 5/9 in females. The difference in the incidence of lung tumors between control and DAH is not statistically significant but difference between control and MAH group is significant. Tumor types observed were adenoma and papillary adenocarcinoma. Some of these tumors were transplantable. Few liver hemangiomas were observed in control as well as other groups and the difference was not statistically significant and hence not included in tumor incidence. The difference observed between mutagenicity and carcinogenicity of MAH and DAH does not appear to be due to difference in the amount used (different molecular weights) since both were used near or at LD5,, dose in short term tests while in long term studies they were toxic and hence it was difficult to use various doses. TABLE 2 TUMOR INCIDENCE IN SWISS MICE TREATED WITH MAH AND DAH Group
MAH Male
Distilled water control 0.8 mg/day per mouse continuous 1.1 mg/day per mouse continuous 1.1 mg/day per mouse for 8 months only
Female
Combined tumor incidence (%I
Male
Female
2118
3120
O/10
O/10
Combined tumor incidence (%I 5138 (13) o/21 (0)
2118
3120
418
519
7110
-
7110 (70)
3110
-
3110 (30)
6111
17124 (71)
2/11
018
2119 (10)
11/13
5138 (13) 9117 (51)
DAH
240
Thus the preliminary observations record for the first time the mutagenicity/carcinogenicity of MAH which is one of the metabolites of INH in animals and humans. REFERENCES 1 Ames, B.N., McCann, J. and Yamasaki, E. (1975) Methods for detecting carcinogens and mutagens with the Solmonellu/mammalian microsome mutagenicity test. Mutat. Res., 31, 347-364. 2 Balo, J. (1979) Role of hydrazine in carcinogenesis. In: Advances in Cancer Research, Vol. 30, pp. 151-164. Editors: G. Klein and S. Weinhouse. Academic Press, London. 3 Bhalerao, E.B. (1982) Studies on the possible mechanism of isoniazid carcinogenesis, Ph.D. thesis, University of Bombay. 4 El Masri, A.M., Smith, J.N. and Williams, R.T. (1958) Studies in detoxification 75. Further observations on the metabolism of hydrazines of aromatic acids. Biochem. J., 68,587-592. 5 Friedman, M.A. and Staub, J. (1976) Inhibition of mouse testicular DNA synthesis by mutagens and carcinogens as a potential simple mammalian assay for mutagenesis. Mutat. Res., 37,67-76. 6 Goodmann, L.S. and Gilman, A. (1975) The Pharmacological Basis of Therapeutics, 5th edn., MacMillan Publishing Co., New York. 7 IARC (1974) IARC monographs on the evaluation of carcinogenic risk of chemicals to man, Vol. 4, pp. 125-179. International Agency for Research on Cancer, Lyon. 8 Noda, A., Goromaru, T., Matsuyama, K., Sogabe, K., Hsu, K.Y. and Iguchi, S. (1978) Quantitative determination of hydrazines derived from isoniazid in patients. J. Pharm. Dyn., 1, 132-141. 9 Porcellati, G. and Preziosi, P. (1954) Transformazione biologica dell’idrazide dell’acido isonictinico. Enzymologia, 17, 47-54. 10 Salvadeo, A., Villa, G., Segagni, S. and Criscuolo, D. (1979) Long-term study of oxideralazine in hypertensive patients, Arzneim Forsch., 29, 1753-1755. 11 Schenker, E. and Salzman, R. (1979) Bicyclic 3-hydrazino pyridazines with antihypertensive action. Arzneim Forsch., 29, 1835-1843. 12 Schmid, W. (1975) The micronucleus test. Mutat. Res., 31, 9-15. 13 Timbrell, J.A., Wright, J.M. and Baillie, T.A. (1977) Monoacetyl hydrazine as a metabolite of isoniazid in man. Clin. Pharmacol. Ther., 22, 602-608. 14 Timbrell, J.A., Wright, J.M. and Smith, G.M. (1977) Determination of hydrazine metabolites of isoniazid in human urine by gas chromatography. J. Chromatogr., 138, 165-172. 15 Toth, B. (1980) Actual new cancer causing hydrazines, hydrazides and hydrazones. J. Cancer Res. Clin. Oncol., 97, 97-108.