- Email: [email protected]
Chlorambucil carcinogenesis in BALBc mice
115
Cancer Letters, 55 (1990) 115-120 Elsevier Scientific Publishers Ireland Ltd.
Chlorambucil
carcinogenesis
A. Cavalierea, N. Pietropaok, “Institute
Perugia
of
Pathological
University,
Anatomy
Perugia
in BALB/c
P.F. Alberti” and R. Vitalib and
Histology,
Division
Chlorambucil, a drug used in the treatment of neoplastic and non-neoplastic disease, was administered by gauage to BALB/c mice at a
dose of 1.0 mg/kg body wt. 5 times per week for 12 weeks to test its carcinogenicity. The survival was statistically reduced in treated animals of both sexes (P < 0.001). The treatment induced a significant increase in lung tumours (males, P < 0.001; females, P < 0.001) and lymphoreticular system tumours (males P < 0.01; females, P < 0.001) in both sexes and mammary carcinomas in female mice (P < 0.05). These results with other inuestigations reported in literature, suggest that chlorambucil is carcinogenic in laboratory animals, mutagenic and that it could be a potential carcinogenic hazard to man.
Keywords: chlorambucil; carcinogenesis
antineoplastic
drug;
Introduction Chlorambucil, an antineoplastic drug, has been used to treat patients with Hodgkin’s and Correspondence
to: Dr. Antonio
Cavaliere,
ogical Anatomy
and Histology,
Perugia
0304-3835/90/$03.50
0
Research
and
bDepartment
of Hygiene,
Institute of PatholUniversity,
Box 54,
non-Hodgkin’s lymphomas, chronic lymphocytic leukaemia, Kaposi’s disease and carcinoma of the breast, lung, testis and ovary. It is also used as an immunosuppressive agent in the treatment of systemic lupus erythematosus, rheumatoid arthritis, acute and chronic glomerulonephritis, nephrotic syndrome and Wegener’s granulomatosis. The drug is administered orally in doses of 0.1-0.2 mg/kg body wt. daily for at least 3-6 weeks. In the intermittent oral treatment doses of lo-20 mg daily are given for 2 weeks with rest periods of 2-4 weeks [3,24,26]. Side effects such as bone marrow hypoplasia, gastrointestinal disorders and hepatotoxicity are usually mild and rapidly-reversible though it is possible to induce serious bone marrow hypoplasia with high doses of the drug administered over long periods [3]. Long-term carcinogenesis tests done on mice and rats suggest that chlorambucil is carcinogenic. However in these experiments the chlorambucil was always administered intraperitoneally to the mice. This method is not used when administering the drug to humans. For this reason and since there is not data available for ascertaining the carcinogenicity of this drug on mice by oral administration and since this chemical is suspected of being carcinogenic in man [2,6,7] we decided to investigate its effect in BALB/c mice by oral administration.
1990 Elsevier Scientific Publishers Ireland Ltd.
Published and Printed in Ireland
Cancer
1990)
Summary
Perugia. Italy.
of
(Italy)
(Received 27 June 1990) (Revision received 18 September (Accepted 25 September 1990)
06100
mice
116
Materials and Methods
embedded in paraffin wax and routinely stained with haematoxilyn and eosin. Special stains were used only when necessary. The logrank test [lo] was used to analyse survival rates and the single tumour incidene trend calculated allowing for longevity [ 111.
Two groups of &week-old intact virgin male and female BALB/c/Cb/Se mice bred at the Division of Cancer Research, Institute of Pathological Anatomy, Perugia University, Italy, were used. Group 1: 53 males and 54 female mice received perorally by gavage 1.0 mg/kg body wt. chlorambucil in aqueous suspension 5 times per week for 12 weeks. Group 2: 50 male and 50 female control mice received only sterile saline solution. The chlorambucil (99.0% purity) was purchased from Sigma Chemical Co., St Louis, MO, U.S.A. and stored at - 20°C in the dark. All mice were housed 4-5 to a metal cage, maintained under identical environmental conditions and given water and commercial pellets (Laboratorio Dottori Piccioni. Brescia, Italy) ad libitum. During the experiment, both treated and control animals were inspected daily, killed if moribund and immediately autopsied. All tumours and pathologically altered organs were removed, fixed in 10% buffered formalin,
x of swvivom rm .-_ ____Lr_______, *. **.* . 1’. . . !, 90-
eo-
m-
A \ -
L-=.\ \,
Results
I and II give types, incidence of tumours and latency periods.
Lymphoreticular system
The rise in tumour incidence was statistically significant in both sexes (males P < 0.01; females P < 0.001). By the Pattengale and Taylor histological classification [9] the majority of tumours were follicular center ceil lymphomas and granulocytic leukaemias. Some
\\ , ‘?+,, .. . \ . ..
\
_-__--
ago
Fig. 1.
Survival of chlorambucil-treated and control mice.
None
‘One squamous cell bOne squamous cell of the ovary. cone squamous cell dOne haemangioma
2
50 50
53 54
No.
Mice
M F
M F
Sex
and site of tumours
22 12
49 50
No.
carcinoma of the forestomach. of the liver.
5 4
7 24
No.
10.0 8.0
13.2 44.4
%
of the liver; 1 haemangiosarcoma
44.0 24.0
92.4 92.5
46
38.0 14.0
88.6 85.1
of subcutaneous
19 7
47 46
No.
%
Lymphoreticular system
mice
Lung
Number of animals with tumor of:
Tumourbearing
in treated and control mice.
carcinoma of the skin. papilloma of the skin; 1 haemangioma
Chlorambucil 1 mg/kg
1
b.w.
Treatment
Incidence
Group No.
Table 1.
connective
0 1
0 4
No.
1’ Id
la 4b
No.
Other tissues
tissue; 1 granulosa-cell
0.0 2.0
0.0 7.4
%
Mammary gland
tumour
2.0 2.0
1.8 7.4
%
118
Table II.
Mean latency periods (L.P.) and range in weeks of tumours in treated and control mice.
Mice
Lymphoreticular
M F M F
Treated Control
system
Mean L.P.
Range
Mean L.P.
Range
Mean L.P.
Range
48.7 49.5 83.2 87.5
17-67 9-66 71-97 71-101
55.6 52.3 80.2 98.5
22-67 18-66 48-97 78-110
0.0 47.0 0.0 95.0
0 34-54 0 95
small lymphocytic lymphomas and lymphoplasmocytoid lymphomas were also seen. Lung Statistical analysis showed that lung tumour incidence was highly significant in both sexes (P < 0.001). Histologically the lung tumours were adenomas composed of columnar or cuboid cells with scarce weakly-acidophilic cytoplasm and round or oval nuclei arranged in papillary, pseudoglandular and cord-like structure. Mammary
Mammary gland
Lung
gland
Four tumours, 3 adenocarcinomas type B and 1 adenoacanthoma [ 181 were found in treated female mice. The only adenocarcinoma type B observed in control mice was found in a 103-week-old female mouse. The difference between treated and control animals was statistically significant (P < 0.05). Discussion To our knowledge only 3 previous longterm experiments in mice have been carried out using chlorambucil which never was administered orally, the route of choice in man. Salaman and Roe [15] observed an initiating effect in a two-stage skin carcinogenesis experiment in 5 strain mice. Shimkin et al. [16] reported an increased incidence of lung tumours in A/J mice after i.p. injections of chlorambucil3 times per week for 4 weeks at a total dose of 37.11, 149.97 and 419.79 mg/kg
body wt. but not at 9.55 mg/kg body wt. These data are difficult to evaluate since there are no exact numbers for division into subgroups as to sex of treated animals. Weisburger et al. [25] observed a statistically significant increased incidence of lung tumours and a probable rise in tumour incidence of lymphoretitular system and ovaries in Swisswebsterderived mice who had received i.p. injections of 3 and 1.5 mg/kg body wt. chlorambucil 3 times per week for 6 months. The present study demonstrates that also oral administration of chlorambucil induces an highly significant increase in lung tumours (males, P < 0.001; females 0.001) as in the Shimkin et al. and Weisburger et al. experiments [16,25]. Furthermore our experiment show that the drug also provokes a statistically significant rise in tumour incidence of lymphoreticular system in both sexes (males, P < 0.01; females, P < 0.001) and mammary carcinomas in female mice (P < 0.05). Long-term tests on Sprague-Dawley rats also indicate that chlorambucil after i.p. injections induces a significant increase of the lymphoreticular system tumours in males, [25] and when administered by oral gavage in female rats induced tumours of mammary glands, central and peripheral nervous system, hematopoietic and lymphatic system and external auditory canal [ 11. Short-term tests indicate that chlorambucil is mutagenic in Escherichia coli Sd-4-73 [20], in the diploid strain D4 of Saccharomyces cerevisiae [27] and in the haploid wild-type strain of Saccharomyces cerevisiae [ 141. Chromosomal
119
aberration have been observed in short-term cultures of human peripheral lymphocytes following exposure to chlorambucil in vitro [ 12,191; chromosomal changes have been. also found in rat embryos 6-30 h after i.p. injections of 8 mg/kg body wt. chlorambucil on the 13th day of pregnancy [17]. Finally a small increase in chromosomal damage has been found in bone-marrow from patients receiving chlorambucil for chronic lymphocytic leukaemia or in peripheral blood lymphocytes from patients treated with chlorambucil for non-neoplastic diseases [ 13,191. The literature includes numerous reports of neoplasms occurring in patients after administration of chlorambucil both for neoplastic and non-neoplastic illness therapy [4,5,8,21-231. This has also been confirmed by the only available randomized study carried out on 431 patients suffering from polycythemia Vera. This study reported a 13-fold increase in the incidence of acute non-lymphocytic leukaemia in patients treated with chlorambucil. This increase was 2.3 times higher with respect to patients treated with radioactive phosphorous 121. In conclusion the results of the present study, together with experimental and human pathology data in literature suggest that chlorambucil is mutagenic, carcinogenic in laboratory animals (mouse, rat) and that as regards man there ‘is a serious oncogenic risk connected with the use of this chemical.
A., Donovan, P.B., Ellis, J.T., Landaw, S.A., Laszlo, J., Najean, Y., Pisciotta, A.V. and Wasserman, L.R. (1981) Increased incidence of acute leukemia in polycythemia vera associated with chlorambucil therapy. N. Engl. J.
4
Med., 304,441-447. Calabresi, P. and Parks, R.E. Jr. (1985) Anttproliferative agents and drugs used for immunosupression. In: Goodman and Gilman’s The Pharmacological Basis of Therapeutics, pp. 1247-1306. Editors: A. Goodman Gilman, L.S. Goodman, T.W. Rall and F. Murad. MacMillan Publishing Co., New York. Carcassone, Y., Gastuat, J.A. and Blanc, A.P. (1978)
5
Acute leukemia after prolonged immunosupressive therapy for glomerulonephritis. Cancer Treat. Rep., 62, 1110 -1111. Fiere, D., Felman, P., Vu Van, H. and Colffier, B. (1978)
3
6
7
8
Chemicals to Humans. Some Antineoplastic and Immunosupressive Agents, Vol. 26, pp. 115-136, Lyon, France. International Agency for Research on Cancer (1981) IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans. Overall Evaluation icity: An Updating of lARC Monographs Suppl. 7, pp. 144-145, Lyon, France. Lebranchu, Y., Druker. J., Nivet, H.,
of CarcinogenVol. 1 to 42. Rolland,
J.C.,
Grenier, B., Lejars, O., Lamagnere, J.P. and Buriot, D. (1980) Acute monoblastic leukemia in child receiving chlorambucil for juvenile rhematoid arthritis. Lancet, i, 649. 9
10
Acknowledgements 11
This work was supported by a 40% grant from the Minister0 della Pubblica Istruzione, Italy, 1985, for the Institute of Pathological Anatomy and Histology, 1st Chair.
Pattengale, P.K. and Taylor, C.R. (1983) Experimental models of lymphoproliferative disease. The mouse as a model for human non Hodgkin’s lymphomas and related leukemias. Am. J. Pathol., 113.237-265. Peto, R., Pike, M.C., An&age, P., Breslow, N.E., Cox, D.R., Howard, S.V., Mantel, N., McPherson, K., Peto, J. and Smith. P.G. (1977) Design and analysis of randomized clinical trials requiring prolonged observation of each patient. II. Analysis and examples. Br. J. Cancer, 35, l39. Peto, R., Pike, M.C., Day, N.E., Gray, R.G., Lee, P.N., Parish, S., Peto, J., Richards, S. and Wahrendorf, J. (1980) Guidelines for simple, sensitive significance tests for carcinogenic effects in long-term animal experiments. IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans. Suppl. 2. Long-term and
References 12 Berger, M.R., Habs. M. and Schmahl, D. (1985) Comparative carcinogenic activity of prednimustine, chlorambucil, prednisolone and chlorambucil plus prednisolone in Sprague-Dawley rats. Arch. Geschwulstforsch., 55 429 -442. Berk, P.D., Goldberg, J.D., Silverstein, M.N., Weinfeld,
Acute myeloid leukemia after administration of chlorambucil. Two cases. Nouv. Presse Mkd., 7,756. International Agency for Research on Cancer (1981) IARC Monographs on the Evaluation of the Carcinogenic Risk of
short-term screening assays for carcinogens: A critical appraisal, pp.311-426, Lyon, France. Reeves, B.R. and Margoles, C. (1974) Preferential location of chlorambucil-induced somes of normal
human
breakage
lymphocytes.
in the Mutat.
chromoRes..
26,
205-208.
13
Reeves,
B.R.,
Pickup,
V.L.
and Lawler,
chromosome
study of patients
chlorambucil.
Br. Med. J., iv, 22-23.
S.D.
(1975)
with uveitis treated
A
with
120
14
15
16
17
18
19
20
Ruhland, A., Fleer, R. and Brendel, M. (1978) Genetic activity of chemicals in yeast: DNA alterations and mutations induced by alkyjating anti-cancer agents. Mutat. Res., 58,241-250. Salaman, M.H. and Roe, F.J.C. (1956) Further tests for tumour-initiating activity: N,N-di(2-chloroethyb-p-aminophenylbutyrtc acid (CB 1348) as an initiator of skin tumour formation in the mouse. Br. J. Cancer, 10,363-378. Shimkin, M.B., Weisburger, J.H., Weisburger, E.K., Gubareff, N. and Suntzeff, V. (1966) Bioassay of 29 alkylating chemicals by the pulmonary-tumor response in strain A mice. J. Natl. Cancer Inst., 36,915-935. Soukup, S., Takacs, E. and Warkany, J. (1967) Chromosome changes in embryos treated with various teratogens. J. Embryol. Exp. Morphol., 18,215-226. Squartini, F. (1979) Tumours of the mammary gland. In: Pathology of Tumours in Laboratory Animals. Vol. II. Tumours of the mouse, pp. 43-90. Editor: V.S. Turusov. IARC Scientific Publications, No. 23, Lyon, France. Stevenson, A.C. and Patel, C. (1973) Effects of chlorambucil on human chromosomes. Mutat. Res., 18, 333351. Szybalski, W. (1958) Special microbiological systems. If. Observations on chemical mutagenesis isms. Ann. N.Y. Acad. Sci., 76,475-489.
in micro-organ-
21
22
23 24
Tchemia, G., Mielot, F., Subtil, E. and Parmentier, C. (1976) Acute myeloblastic leukemia after immunodepressive therapy for primary nonmalignant disease. Blood Cells, 2,67-80. Tulliez, M., Ricard, M.F., Jan, F. and Sultan, C. (1974) Preleukaemic abnormal myelopoiesis induced by chlorambucil: a case study. Stand. J. Haematol., 13, 179-183. Videbaek, A. (1971) Unusual cases of myelomatosis. Br. Med. J., ii, 326. Wade, a. (1977) Martindale, The Extra Pharmacopoeia,
26
27th ed., pp. 131-133, The Pharmaceutical Press, London. Weisburger, J.H., Griswold, D.P., Prejean, J.D., Casey, A.E., Wood, H.B. and Weisburger, E.K. (1975) The carcinogenic properties of some of the principal drugs used in clinical cancer chemotherapy. Recent Results Cancer Res., 52, l-17. Williams, S.A., Makker, S.P., Ingelfinger, J.R. and
27
Grupe, W.E. (1980) Log-term evaluation of chlorambucil plus prednisone in the idiopathic nephrotic syndrome in childhood. New Engl. J. Med., 302.929-933. Zimmermann, F.K. (1971) Induction of mitotic gene con-
25
version by mutagens.
Mutat. Res., 11, 327-337.
Cancer Letters, 55 (1990) 115-120 Elsevier Scientific Publishers Ireland Ltd.
Chlorambucil
carcinogenesis
A. Cavalierea, N. Pietropaok, “Institute
Perugia
of
Pathological
University,
Anatomy
Perugia
in BALB/c
P.F. Alberti” and R. Vitalib and
Histology,
Division
Chlorambucil, a drug used in the treatment of neoplastic and non-neoplastic disease, was administered by gauage to BALB/c mice at a
dose of 1.0 mg/kg body wt. 5 times per week for 12 weeks to test its carcinogenicity. The survival was statistically reduced in treated animals of both sexes (P < 0.001). The treatment induced a significant increase in lung tumours (males, P < 0.001; females, P < 0.001) and lymphoreticular system tumours (males P < 0.01; females, P < 0.001) in both sexes and mammary carcinomas in female mice (P < 0.05). These results with other inuestigations reported in literature, suggest that chlorambucil is carcinogenic in laboratory animals, mutagenic and that it could be a potential carcinogenic hazard to man.
Keywords: chlorambucil; carcinogenesis
antineoplastic
drug;
Introduction Chlorambucil, an antineoplastic drug, has been used to treat patients with Hodgkin’s and Correspondence
to: Dr. Antonio
Cavaliere,
ogical Anatomy
and Histology,
Perugia
0304-3835/90/$03.50
0
Research
and
bDepartment
of Hygiene,
Institute of PatholUniversity,
Box 54,
non-Hodgkin’s lymphomas, chronic lymphocytic leukaemia, Kaposi’s disease and carcinoma of the breast, lung, testis and ovary. It is also used as an immunosuppressive agent in the treatment of systemic lupus erythematosus, rheumatoid arthritis, acute and chronic glomerulonephritis, nephrotic syndrome and Wegener’s granulomatosis. The drug is administered orally in doses of 0.1-0.2 mg/kg body wt. daily for at least 3-6 weeks. In the intermittent oral treatment doses of lo-20 mg daily are given for 2 weeks with rest periods of 2-4 weeks [3,24,26]. Side effects such as bone marrow hypoplasia, gastrointestinal disorders and hepatotoxicity are usually mild and rapidly-reversible though it is possible to induce serious bone marrow hypoplasia with high doses of the drug administered over long periods [3]. Long-term carcinogenesis tests done on mice and rats suggest that chlorambucil is carcinogenic. However in these experiments the chlorambucil was always administered intraperitoneally to the mice. This method is not used when administering the drug to humans. For this reason and since there is not data available for ascertaining the carcinogenicity of this drug on mice by oral administration and since this chemical is suspected of being carcinogenic in man [2,6,7] we decided to investigate its effect in BALB/c mice by oral administration.
1990 Elsevier Scientific Publishers Ireland Ltd.
Published and Printed in Ireland
Cancer
1990)
Summary
Perugia. Italy.
of
(Italy)
(Received 27 June 1990) (Revision received 18 September (Accepted 25 September 1990)
06100
mice
116
Materials and Methods
embedded in paraffin wax and routinely stained with haematoxilyn and eosin. Special stains were used only when necessary. The logrank test [lo] was used to analyse survival rates and the single tumour incidene trend calculated allowing for longevity [ 111.
Two groups of &week-old intact virgin male and female BALB/c/Cb/Se mice bred at the Division of Cancer Research, Institute of Pathological Anatomy, Perugia University, Italy, were used. Group 1: 53 males and 54 female mice received perorally by gavage 1.0 mg/kg body wt. chlorambucil in aqueous suspension 5 times per week for 12 weeks. Group 2: 50 male and 50 female control mice received only sterile saline solution. The chlorambucil (99.0% purity) was purchased from Sigma Chemical Co., St Louis, MO, U.S.A. and stored at - 20°C in the dark. All mice were housed 4-5 to a metal cage, maintained under identical environmental conditions and given water and commercial pellets (Laboratorio Dottori Piccioni. Brescia, Italy) ad libitum. During the experiment, both treated and control animals were inspected daily, killed if moribund and immediately autopsied. All tumours and pathologically altered organs were removed, fixed in 10% buffered formalin,
x of swvivom rm .-_ ____Lr_______, *. **.* . 1’. . . !, 90-
eo-
m-
A \ -
L-=.\ \,
Results
I and II give types, incidence of tumours and latency periods.
Lymphoreticular system
The rise in tumour incidence was statistically significant in both sexes (males P < 0.01; females P < 0.001). By the Pattengale and Taylor histological classification [9] the majority of tumours were follicular center ceil lymphomas and granulocytic leukaemias. Some
\\ , ‘?+,, .. . \ . ..
\
_-__--
ago
Fig. 1.
Survival of chlorambucil-treated and control mice.
None
‘One squamous cell bOne squamous cell of the ovary. cone squamous cell dOne haemangioma
2
50 50
53 54
No.
Mice
M F
M F
Sex
and site of tumours
22 12
49 50
No.
carcinoma of the forestomach. of the liver.
5 4
7 24
No.
10.0 8.0
13.2 44.4
%
of the liver; 1 haemangiosarcoma
44.0 24.0
92.4 92.5
46
38.0 14.0
88.6 85.1
of subcutaneous
19 7
47 46
No.
%
Lymphoreticular system
mice
Lung
Number of animals with tumor of:
Tumourbearing
in treated and control mice.
carcinoma of the skin. papilloma of the skin; 1 haemangioma
Chlorambucil 1 mg/kg
1
b.w.
Treatment
Incidence
Group No.
Table 1.
connective
0 1
0 4
No.
1’ Id
la 4b
No.
Other tissues
tissue; 1 granulosa-cell
0.0 2.0
0.0 7.4
%
Mammary gland
tumour
2.0 2.0
1.8 7.4
%
118
Table II.
Mean latency periods (L.P.) and range in weeks of tumours in treated and control mice.
Mice
Lymphoreticular
M F M F
Treated Control
system
Mean L.P.
Range
Mean L.P.
Range
Mean L.P.
Range
48.7 49.5 83.2 87.5
17-67 9-66 71-97 71-101
55.6 52.3 80.2 98.5
22-67 18-66 48-97 78-110
0.0 47.0 0.0 95.0
0 34-54 0 95
small lymphocytic lymphomas and lymphoplasmocytoid lymphomas were also seen. Lung Statistical analysis showed that lung tumour incidence was highly significant in both sexes (P < 0.001). Histologically the lung tumours were adenomas composed of columnar or cuboid cells with scarce weakly-acidophilic cytoplasm and round or oval nuclei arranged in papillary, pseudoglandular and cord-like structure. Mammary
Mammary gland
Lung
gland
Four tumours, 3 adenocarcinomas type B and 1 adenoacanthoma [ 181 were found in treated female mice. The only adenocarcinoma type B observed in control mice was found in a 103-week-old female mouse. The difference between treated and control animals was statistically significant (P < 0.05). Discussion To our knowledge only 3 previous longterm experiments in mice have been carried out using chlorambucil which never was administered orally, the route of choice in man. Salaman and Roe [15] observed an initiating effect in a two-stage skin carcinogenesis experiment in 5 strain mice. Shimkin et al. [16] reported an increased incidence of lung tumours in A/J mice after i.p. injections of chlorambucil3 times per week for 4 weeks at a total dose of 37.11, 149.97 and 419.79 mg/kg
body wt. but not at 9.55 mg/kg body wt. These data are difficult to evaluate since there are no exact numbers for division into subgroups as to sex of treated animals. Weisburger et al. [25] observed a statistically significant increased incidence of lung tumours and a probable rise in tumour incidence of lymphoretitular system and ovaries in Swisswebsterderived mice who had received i.p. injections of 3 and 1.5 mg/kg body wt. chlorambucil 3 times per week for 6 months. The present study demonstrates that also oral administration of chlorambucil induces an highly significant increase in lung tumours (males, P < 0.001; females 0.001) as in the Shimkin et al. and Weisburger et al. experiments [16,25]. Furthermore our experiment show that the drug also provokes a statistically significant rise in tumour incidence of lymphoreticular system in both sexes (males, P < 0.01; females, P < 0.001) and mammary carcinomas in female mice (P < 0.05). Long-term tests on Sprague-Dawley rats also indicate that chlorambucil after i.p. injections induces a significant increase of the lymphoreticular system tumours in males, [25] and when administered by oral gavage in female rats induced tumours of mammary glands, central and peripheral nervous system, hematopoietic and lymphatic system and external auditory canal [ 11. Short-term tests indicate that chlorambucil is mutagenic in Escherichia coli Sd-4-73 [20], in the diploid strain D4 of Saccharomyces cerevisiae [27] and in the haploid wild-type strain of Saccharomyces cerevisiae [ 141. Chromosomal
119
aberration have been observed in short-term cultures of human peripheral lymphocytes following exposure to chlorambucil in vitro [ 12,191; chromosomal changes have been. also found in rat embryos 6-30 h after i.p. injections of 8 mg/kg body wt. chlorambucil on the 13th day of pregnancy [17]. Finally a small increase in chromosomal damage has been found in bone-marrow from patients receiving chlorambucil for chronic lymphocytic leukaemia or in peripheral blood lymphocytes from patients treated with chlorambucil for non-neoplastic diseases [ 13,191. The literature includes numerous reports of neoplasms occurring in patients after administration of chlorambucil both for neoplastic and non-neoplastic illness therapy [4,5,8,21-231. This has also been confirmed by the only available randomized study carried out on 431 patients suffering from polycythemia Vera. This study reported a 13-fold increase in the incidence of acute non-lymphocytic leukaemia in patients treated with chlorambucil. This increase was 2.3 times higher with respect to patients treated with radioactive phosphorous 121. In conclusion the results of the present study, together with experimental and human pathology data in literature suggest that chlorambucil is mutagenic, carcinogenic in laboratory animals (mouse, rat) and that as regards man there ‘is a serious oncogenic risk connected with the use of this chemical.
A., Donovan, P.B., Ellis, J.T., Landaw, S.A., Laszlo, J., Najean, Y., Pisciotta, A.V. and Wasserman, L.R. (1981) Increased incidence of acute leukemia in polycythemia vera associated with chlorambucil therapy. N. Engl. J.
4
Med., 304,441-447. Calabresi, P. and Parks, R.E. Jr. (1985) Anttproliferative agents and drugs used for immunosupression. In: Goodman and Gilman’s The Pharmacological Basis of Therapeutics, pp. 1247-1306. Editors: A. Goodman Gilman, L.S. Goodman, T.W. Rall and F. Murad. MacMillan Publishing Co., New York. Carcassone, Y., Gastuat, J.A. and Blanc, A.P. (1978)
5
Acute leukemia after prolonged immunosupressive therapy for glomerulonephritis. Cancer Treat. Rep., 62, 1110 -1111. Fiere, D., Felman, P., Vu Van, H. and Colffier, B. (1978)
3
6
7
8
Chemicals to Humans. Some Antineoplastic and Immunosupressive Agents, Vol. 26, pp. 115-136, Lyon, France. International Agency for Research on Cancer (1981) IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans. Overall Evaluation icity: An Updating of lARC Monographs Suppl. 7, pp. 144-145, Lyon, France. Lebranchu, Y., Druker. J., Nivet, H.,
of CarcinogenVol. 1 to 42. Rolland,
J.C.,
Grenier, B., Lejars, O., Lamagnere, J.P. and Buriot, D. (1980) Acute monoblastic leukemia in child receiving chlorambucil for juvenile rhematoid arthritis. Lancet, i, 649. 9
10
Acknowledgements 11
This work was supported by a 40% grant from the Minister0 della Pubblica Istruzione, Italy, 1985, for the Institute of Pathological Anatomy and Histology, 1st Chair.
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