Fish Analysis for Drug and Chemicals Mediated Cellular
Toxicity
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Ajit Kumar Saxena Amit Kumar
Department of Pathology BioAxis DNA Research Centre Private Ltd.
and Laboratory Medicine Hyderabad, Telangana, India
All India Institute of Medical Sciences
Patna, Bihar, India
ISSN 2191-530X ISSN 2191-5318 (electronic)
SpringerBriefs in Applied Sciences and Technology
ISBN 978-981-15-4699-0 ISBN 978-981-15-4700-3 (eBook)
https://doi.org/10.1007/978-981-15-4700-3
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Contents
1 Introduction of Cyclophosphamide . . . . . . . . . . . . . . . . . . . . . . . . . . 1
1.1 Structure of Cyclophosphamide . . . . . . . . . . . . . . . . . . . . . . . . . . 1
1.2 Activation of Cyclophosphamide . . . . . . . . . . . . . . . . . . . . . . . . . 2
1.3 Risk Factors of Cyclophosphamide . . . . . . . . . . . . . . . . . . . . . . . 3
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
2 Effect of Cyclophosphamide on Chromosomes . . . . . . . . . . . . . . . . . 7
2.1 Experimental Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
2.2 Interpretation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
3 Effect of Cyclophosphamide on Testis—Protein Profile Study . . . . . 25
3.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
3.2 Interpretation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
4 Effect of Cyclophosphamide on Testis—A Histological Study . . . . . . 43
4.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
4.2 Experimental Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
4.2.1 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
4.3 Findings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
4.4 Tunica Albugenia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
4.5 Interpretation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
5 Effect of Arsenic Exposure in Reproductive Health . . . . . . . . . . . . . 59
5.1 Experimental Design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
5.1.1 Arsenic and Male Reproduction . . . . . . . . . . . . . . . . . . . . 60
5.1.2 Arsenic and Female Reproduction . . . . . . . . . . . . . . . . . . 70
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
v
vi Contents
6 Fish, Genetic and Cellular Toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . 81
6.1 Cytotoxic Agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82
6.2 Factors and Mechanism of Cell Toxicity . . . . . . . . . . . . . . . . . . . 84
6.3 Biomarkers in Cell Toxicity Study . . . . . . . . . . . . . . . . . . . . . . . . 85
6.4 Measuring the Cell Toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
About the Authors
Prof. Ajit Kumar Saxena has received his Ph.D. from
the Department of Anatomy in Cyotgenetics from the
Institute of Medical Sciences, BHU, in the year of
1990. During his Ph.D., he studied how anticancer
drugs cause interference with normal development
of the fetus and with special reference to the cause of
abnormality during differentiation of male gonads
which lead to infertility including congenital anomalies.
After receiving his Ph.D. degree, he joined NIH-funded
Indo-US project at AIIMS New Delhi, where he
identified the role of novel antigen IL6 as a signal
traducing agent in human glioblastoma. He also worked
in various renowned institutions of India and abroad
including CCMB, CDRI, A.M.C., B.P.K.I.H.S.
(Dharan, Nepal) and BHU, Varanasi. Currently, he is
working as Professor and Head in the Department of
Pathology/Lab Medicine specialized in clinical genetics
in All India Institute of Medical Sciences Patna. He also
possess administrative responsibilities as Head of the
Department in prestigious institutions of India and
abroad. In addition, he has also served his duties of
Scientific Advisor to the government. His research
interest includes study of gene polymorphism in
congenital neural tube defects/tumor biology/infertility
and cytokine gene expression in chronic wounds and
hypertrophic scars. Currently, his research interest
includes role of stem cells in translational research.
Of course, he published more than 100 articles in
peer-reviewed journals of international reputes with
high citations. Based on his research and fellowship
training, he has received several awards and honors,
vii
viii About the Authors
such as Gold Medal Award, Confer ‘Vivek Ratan
Award’ and Millennium Award, Health Care Excellence
Award and Life Time Achievement Awards (three
times). Besides his research, teaching and administra-
tion, he has also guided postgraduate (Ph.D., MD, MS,
M.Tech.) students. He is serving as Editorial Member of
various reputed journals and Reviewer for several
government-funded projects and research papers. He
also acquired and completed several government-
funded research projects (ICMR, DBT, CSIR, DST).
He is ‘Fellow’ and ‘Member’ of various scientific
organizations, National Academy of Sciences, USA,
and National Academy for Advancement of Science and
Technology, USA, and Member of National Academy
of Medical Sciences, India. He is also Fellow of Indian
Association of Biomedical Scientist, Chennai, and
Fellow of Indian Association of Applied Biotech-
nology, Mysore. Currently, he is engaged in the
developing Advanced Clinical Genetics Laboratory for
Diagnostic, Training and Research in India (Bihar).
Dr. Amit Kumar is a DNA Forensics Professional,
Entrepreneur, Engineer, Bioinformatician and an IEEE
Volunteer. In 2005 he founded first Private DNA
Testing Company Bio Axis DNA Research Centre
(P) Ltd in Hyderabad, India with an US Collaborator.
He has vast experience of training 1000+ Crime
investigation officers and helped 750+ Criminal and
non-criminal cases to reach justice by offering analyt-
ical services in his laboratory. His group also works
extensively on Genetic Predisposition risk studies of
cancers and has been helping many cancer patients
from 2012 to fight and win the battle against cancer.
Amit was member of IEEE Strategy Development and
Environmental Assessment committee (SDEA) of
IEEEMGA. He is senior member of IEEE and has
been a very active IEEE Volunteer at Section, Council,
Region, Technical Societies of Computational Intelli-
gence and Engineering in Medicine and Biology and at
IEEE MGA levels in several capacities. He has driven
number of IEEE Conferences, Conference leadership
programs, Entrepreneurship development workshops,
Innovation and Internship related events. Currently He
is a Professor at SJB Institute of Technology Bangalore,
IEEE MGA Nominations and Appointments committee
member and Chairman of IEEE Hyderabad Section.
Chapter 1
Introduction of Cyclophosphamide
Recent epidemiological investigation in early pregnancy has revealed an increased
embryonic loss in nurses occupationally exposed to antineoplastic drugs, during the
first trimester of pregnancy [1]. Drug treatment often in pregnant female has been
associated with a number of malformations in both animals [2, 3] and humans [4].
There has been also increasing number of reports of gonadal damage following
cytotoxic drug therapy for the treatment of malignant and non-malignant conditions
[5].
Drugs in the alkylating agent category used in cancer therapy, such as cyclophos-
phamide (CP), have also been anecdotally associated with gonadal damage in anoma-
lies. Thus, the interest in the possible use of CP in therapeutic areas other than cancer
chemotherapy has increased concern regarding the effects on reproductive processes
[6]. In human pregnancy, it has been particularly difficult to obtain data on the action
of drugs and environmental chemicals [7]. The use for an animal study in this direc-
tion therefore becomes imperative. Interestingly, what effect different doses of CP
have on the male gonad of the offspring is exposed antenatally to CP during the
critical period of gonadal development.
1.1 Structure of Cyclophosphamide
CP is a potent cytostatic agent related to the class of compounds known as alky-
lating agents. CP together with a number of related N-phosphorylated derivatives of
nitrogen mustard is an attempt to obtain a compound with greater antitumor speci-
ficity than found in conventional alkylating agents of the nitrogen mustard class.
Endoxan-Asta brand of cyclophosphamide is chemically N, N-bis (B-chloroethyl)-
N-O-propylenephosphoric acid ester diamide monohydrate (Fig. 1.1) to release the
active polyfunctional alkylating form.
© The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2020 1
A. K. Saxena and A. Kumar, Fish Analysis for Drug and Chemicals Mediated Cellular
Toxicity, SpringerBriefs in Applied Sciences and Technology,
https://doi.org/10.1007/978-981-15-4700-3_1
2 1 Introduction of Cyclophosphamide
Fig. 1.1 Cyclophosphamide is a synthetic alkylating agent chemically related to the nitrogen
mustards with antineoplastic and immunosuppressive activities. In the liver, cyclophosphamide
is converted to the active metabolites aldophosphamide and phosphoramide mustard, which bind
to DNA, thereby inhibiting DNA replication and initiating cell death, MW: 261.08 g/mol
1.2 Activation of Cyclophosphamide
The activity of CP in experimental and clinical cancer was demonstrated and now well
established that the effectiveness of CP does not arise from enzymatic cleavage by
tumor cells as originally proposed. This compound is activated by hepatic microsomal
enzymes, with the active metabolites reaching their target site through the systemic
circulation. The complex mode of enzymes with the active metabolites is reaching
their target site through the systemic circulation. The complex mode of activation
of CP and the identity of the active metabolites have been the subject of intense
study by several groups of investigators. The current view of activation [8, 11],
which may well undergo full further modification, is that the liver cytochrome p-
450 microsomal mixed functional oxidase system converts cyclophosphamide to
4-hydroxycyclophospamide, within equilibrium with its acyclic tautomeric form,
aldophosphamide.
Cyclophosphamide further oxidation of the latter compound possibly through
the mediation of liver aldehyde oxidase and the other aldehyde-metabolizing
enzymes results in the formation of known metabolites, carboxyphosphamide and
4-ketocyclophoasphamide which are relatively non-toxic. The cleavage of aldophos-
phamide by a ß-elimination reaction yields phosphoramide mustard and acrolin,
both of which are highly cytotoxic and may represent the active form of the drug [9].
Although both of these compounds have now been isolated as products of cyclophos-
phamide activation [10, 11], it has not been established that they are generated
in sufficient quantity to account for the total cytotoxic activity. Hydrolysis of P-N
linkage of the parent compound or of its metabolites could yield the alkylating agent
non-nitrogen mustard, also an end product of cyclophasphamide metabolites. The
1.2 Activation of Cyclophosphamide 3
cytotoxic activity of this compound appears to be too low to contribute appreciably
to the total pharmacologic activity of cyclophosphamide.
1.3 Risk Factors of Cyclophosphamide
CP is mutagenic [3], carcinogenic [12] and teratogenic [3, 18] in nature [13]. CP
is a first substance known to induce chromosome rearrangements and gene muta-
tion in germ cells of experimental animals [14]. The mutagenic activity has been
reported for a variety of mammalian (including human) somatic cells and for germ
cells of experimental mammals [14]. An increased number of chromosomal aber-
rations were observed in the peripheral blood lymphocytes of children treated with
3–5 mg/day of CP for 6–8 months for non-malignant conditions. Treatment with CP
has been associated with the production of secondary cancer, significant incidence
of bladder carcinoma, non-Hodgkin’s lymphoma and squamous cell carcinoma of
the skin which have been observed in patients treated for non-malignant diseases,
rheumatoid arthritis and glomerulonephritis [15]. The incidence of a second cancer
also increased in CP-treated cancer patients. These malignancies have been bladder
cancer, acute leukemias and reticulum cell sarcomas in some unusual locations [16].
Teratogenic potential of CP in humans has been difficult to evaluate since there are
no epidemiologic data to correctly assess the embryologic risk in man [16]. However,
CP has been reported to cause gonadal damage (oligozoospermia, azoospermia and
amenorrhoea) which may be irreversible in some instances [17]. Also patholog-
ically in man, marked aplasia of sertoli’s cells and loss of germinal epithelium,
lining the seminiferous tubules are reported to be the striking features. In woman,
ovarian atrophy, fibrosis and complete absence of follicular structures have further
been observed to be the primary histologic features. Several reports have shown
that high doses of CP on specific critical days of pregnancy can be teratogenic to
experimental animals [18]. It has been suggested that CP is about three times more
lethal to the fetus than the adult rats [19]. Large doses of CP have been reported to
cause alopecia, nausea and vomiting [20]; fetal cardiomyopathy, hematologic effects,
pulmonary toxicity [21]; hepatotoxicity [22]; mucosal ulceration, skin pigmentation
and anaphylaxis. Transient cerebral dysfunction has also been associated with CP
therapy.
CP has also been used an immunosuppressive agent following organ transplanta-
tion experiments [23]. It caused significant depression of antibody production with
number of antigens [24]. CP was found to be more effective against rapidly prolif-
erative cells. Since CP is more effective against proliferative (in cycle) than non-
proliferative (out of cycle) cells, it was grouped ‘cell cycle-specific’ antineoplastic
agents [25]. The cell cycle specificity of CP appears to be prime determinant in
killing of immunologically competent cells. Embryo transplantation experiments
showed that early CP treatment interfered with the subsequent development of both
the embryo and the mother [26].
4 1 Introduction of Cyclophosphamide
Objectives of study are based on existing knowledge in literature where CP has
been used to see its effect on the adults, especially during spermatogenesis [14, 27–
30]. Despite the fact that CP has been associated with gonadal damage in several
reports, its action during differentiation and development of the testes has failed to
receive even cursory attempt. It is undoubtedly to know the changes in testicular
tissue collected from 1-day-old pups delivered to mother rats exposed to single dose,
2 or 10 or 20 mg/kg of CP on 12th or 15th or 18th day of gestational age to evaluate
the genotoxicity in terms of chromosomal abnormalities and to compare the same in
other organ such as liver and bone-marrow, protein profile alterations, and cellular
changes based on histopathology. Besides this apart from the major objective, it was
also proposed to evaluate the frequency of chromosomal aberrations in embryonic
tissue and/or liver and testes at various intervals (14th, 16th, 18th and 22nd day
of gestation), after single dose (20 mg/kg) of CP at 12th day of gestation. Further
extended the study of the protein profile changes in normal developing embryos
between 12th and 15th day of gestation, critical period when the embryos were
insulted as a part of the major objective in this study, and to look into the reproductive
performance (by breeding/mating experiment) followed by looking into the protein
profile, in case infertility was induced, of the adult males and females from the
following experiments where mother rats were exposed to single dose of CP of 2
or 10 or 20 mg/kg at 12th or 15th or 18th day of gestation, or a continuous dose
(5 mg/kg) of CP on 12th to 15th day of gestation.
References
1. S.G. Selevan, M.L. Lindbohm, R.W. Hornung, K. Hemminki, A study of occupational exposure
to antineoplastic drugs and fetal loss in nurses. N. Engl. J. Med. 313(19), 1173–1178 (1985)
2. J.E. Gibson, B.A. Becker, The teratogenicity of cyclophosphamide in mice. Cancer Res. 28(3),
475–480 (1968)
3. B.F. Hales, Modification of the mutagenicity and teratogenicity of cyclophosphamide in rats
with inducers of the cytochromes P-450. Teratology 24(1), 1–11 (1981)
4. L.H. Greenberg, K.R. Tanaka, Congenital anomalies probably induced by cyclophosphamide.
JAMA 188, 423–426 (1964)
5. S.M. Shalet, Effects of cancer chemotherapy on gonadal function of patients. Cancer Treat.
Rev. 7(3), 141–152 (1980)
6. J.A. Botta Jr., H.C. Hawkins, J.H. Weikel Jr., Effects of cyclophosphamide on fertility and
general reproductive performance of rats. Toxicol. Appl. Pharmacol. 27(3), 602–611 (1974)
7. R. Vogel, H. Spielmann, Potentiating effect of caffeine on embryotoxicity of cyclophosphamide
treatment in vivo during the preimplantation period. Teratog. Carcinog. Mutagen. 7(2), 169–174
(1987)
8. T.A. Connors, P.J. Cox, P.B. Farmer, A.B. Foster, M. Jarman, Some studies of the active inter-
mediates formed in the microsomal metabolism of cyclophosphamide and isophosphamide.
Biochem. Pharmacol. 23(1), 115–129 (1974)
9. H.L. Gurtoo, S.K. Bansal, Z. Pavelic, R.F. Struck, Effects of the induction of hepatic microsomal
metabolism on the toxicity of cyclophosphamide. Br. J. Cancer 51(1), 67–75 (1985)
10. R.A. Alarcon, J. Meienhofer, Formation of the cytotoxic aldehyde acrolein during in vitro
degradation of cyclophosphamide. Nat. New Biol. 233(42), 250–252 (1971)
References 5
11. M. Colvin, C.A. Padgett, C. Fenselau, A biologically active metabolite of cyclophosphamide.
Cancer Res. 33(4), 915–918 (1973)
12. D. Schmähl, M. Habs, Carcinogenic action of low-dose cyclophosphamide given orally to
Sprague-Dawley rats in a lifetime experiment. Int. J. Cancer 23(5), 706–712 (1979)
13. J.M. Trasler, B.F. Hales, B. Robaire, Chronic low dose cyclophosphamide treatment of adult
male rats: effect on fertility, pregnancy outcome and progeny. Biol. Reprod. 34(2), 275–283
(1986)
14. G.R. Mohn, J. Ellenberger, Genetic effects of cyclophosphamide, ifosfamide and trofosfamide.
Mutat. Res. 32(3–4), 331–360 (1976)
15. L.J. Kinlen, R.N. Hoover, Lymphomas in renal transplant recipients: a search for clustering.
Br. J. Cancer 40(5), 798–801 (1979)
16. A.R. Ahmed, S.M. Hombal, Cyclophosphamide (Cytoxan). A review on relevant pharmacology
and clinical uses. J. Am. Acad. Dermatol. 11(6), 1115–1126 (1984)
17. N. Brock, The oxazaphosphorines. Cancer Treat. Rev. 10(Suppl A), 3–15 (1983)
18. J.E. Gibson, B.A. Becker, Impairment of motor function of neonatal chicks by hemicholinium
treatment during incubation. Toxicol. Appl. Pharmacol. 14(2), 380–392 (1969)
19. S. Chaube, W. Kreis, K. Uchida, M.L. Murphy, The teratogenic effect of 1-beta-D-
arabinofuranosylcytosine in the rat. Protection by deoxycytidine. Biochem. Pharmacol. 17(7),
1213–1216 (1968)
20. J.H. Fetting, L.B. Grochow, M.F. Folstein, D.S. Ettinger, M. Colvin, The course of nausea and
vomiting after high-dose cyclophosphamide. Cancer Treat. Rep. 66(7), 1487–1493 (1982)
21. R.B. Weiss, F.M. Muggia, Cytotoxic drug-induced pulmonary disease: update 1980. Am. J.
Med. 68(2), 259–266 (1980)
22. A.M. Bacon, S.A. Rosenberg, Cyclophosphamide hepatotoxicity in a patient with systemic
lupus erythematosus. Ann. Intern. Med. 97(1), 62–63 (1982)
23. T.E. Starzl, J. Corman, C.G. Groth et al., Personal experience with orthotopic liver transplan-
tation. Transplant. Proc. 4(4), 759–771 (1972)
24. J.L. Turk, D. Parker, Effect of cyclophosphamide on immunological control mechanisms.
Immunol. Rev. 65, 99–113 (1982)
25. F. Valeriote, L. van Putten, Proliferation-dependent cytotoxicity of anticancer agents: a review.
Cancer Res. 35(10), 2619–2630 (1975)
26. R.S. Spielman, J.V. Neel, F.H. Li, Inbreeding estimation from population data: models,
procedures and implications. Genetics 85(2), 355–371 (1977)
27. I.P. Lee, R.L. Dixon, Effects of procarbazine on spermatogenesis determined by velocity sedi-
mentation cell separation technique and serial mating. J. Pharmacol. Exp. Ther. 181(2), 219–226
(1972)
28. K. Fukutani, H. Ishida, M. Shinohara et al., Suppression of spermatogenesis in patients with
Behçet’s disease treated with cyclophosphamide and colchicine. Fertil. Steril. 36(1), 76–80
(1981)
29. F. Pacchierotti, D. Bellincampi, D. Civitareale, Cytogenetic observations, in mouse secondary
spermatocytes, on numerical and structural chromosome aberrations induced by cyclophos-
phamide in various stages of spermatogenesis. Mutat. Res. 119(2), 177–183 (1983)
30. R.L. Schilsky, B.J. Lewis, R.J. Sherins, R.C. Young, Gonadal dysfunction in patients receiving
chemotherapy for cancer. Ann. Intern. Med. 93(1), 109–114 (1980)
Chapter 2
Effect of Cyclophosphamide
on Chromosomes
The chromosome damage has long been recognized as a serious hazard to man
because such a damage is associated with severe clinical disorders [1–3]. Thus, tests
designed to detect the chromosome-damaging potential (clastogenicity) of chemi-
cals have become an established part of the safety evaluation programmes. The best
validated in vivo test for clastogenic potential involves the analysis of either chromo-
somes or micronuclei in rodent bone-marrow cells [4]. The bone marrow is a useful
source because it has proliferating cells with a short cell cycle and is recommended
for estimating the mutagenic activity of chemicals [5, 6].
Cyclophosphamide (CP) is a first substance known to induce chromosome rear-
rangements and gene mutation in germ cells of experimental animals (see [7]). The
clastogenic effect of CP has been assessed in human lymphocyte cultures [6, 8, 9] and
bone-marrow cells. It is also reported to induce chromosomal aberrations and (SCEs)
in human and experimental mammals in vivo [10–17]. Most of the chemical mutagens
induce chromosomal aberrations both in bone-marrow cells and in spermatogonia.
Besides equal or different sensitivities of various tissues [18], spermatogonia seem
to be more sensitive to cell killing than bone-marrow cells. It was observed that
single dose of CP (40 mg, 80 mg or 160 mg/kg) induced structural chromosomal
aberrations including translocation and interfered with the normal development of
bivalents [19]. Organ-specific genotoxicity effect of CP was observed by Ashby and
Beije [20]. CP induced chromosomal aberrations and micronuclei in rat bone-marrow
cells but failed to induce unscheduled DNA synthesis in liver [3, 20]. CP damage in
bone-marrow cells of non-hepatectomized rats was compared with the CP damage
on regenerating liver cells of hepatectomized rats. Results indicated that regenerating
liver cells were more sensitive than bone-marrow cells [21].
Cytogenetic analysis of bone marrow in mice revealed that during the long-term
exposure (7–28 days), the percentage of aberrant cells remained stable but consid-
erable individual variation in the frequency of aberrant cells was observed [5]. The
chromosomal aberration in bone-marrow cells was influenced by the length of the
exposure to mutagen. The frequent aberrations were chromatid and chromosome
breaks. Chromatid exchanges were detected only during the first 24 h after exposure
termination, and chromosome exchange (dicentric and ring chromosome) was seen
© The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2020 7
A. K. Saxena and A. Kumar, Fish Analysis for Drug and Chemicals Mediated Cellular
Toxicity, SpringerBriefs in Applied Sciences and Technology,
https://doi.org/10.1007/978-981-15-4700-3_2
8 2 Effect of Cyclophosphamide on Chromosomes
very rarely [22]. In mice, earlier studies [19, 23–25] showed a significant increase
of chromosomal aberration after a single dose (20, 40 and 60 mg/kg) of CP. The
maximum frequency of aberrant cells occurred 12 and 18 h after 50 mg/kg of CP
application [22], while cytogenetic analysis of cells processed after 24 h yields only
about 60–70% of maximum frequency changes [26–28].
2.1 Experimental Procedure
Rats are putative animal being used for experimental studies. Female Charles Foster
rats of approximately 100–200 days of age weighing about 200–250 g were housed
in wire cages with the males of the same strain (C.F. inbred strain) in the evening,
and pregnancy was confirmed by the presence of sperms in the vaginal smear exam-
ined on the following morning. Sperm-positive day was designated as day ‘zero’ of
pregnancy. Pregnant females were weighed and houses individually in a noise-free
air-conditioned (23 ± 1 °C) animal house maintained on a light dark cycle of 12:12 h.
Pregnant animals were fed on the diet pellet (Hindustan Lever, Bombay, India) and
tap water.
Chemical The chemicals used were cyclophosphamide (Endoxan-Asta) (Khandel-
wal Lab., Bombay, India); colchicine (Bios, India); sodium chloride; potassium chlo-
ride; trisodium citrate; acetic acid; methanol; glycerol; Giemsa powder (B.D.H.,
India); trypsin (Sigma Chemicals, Co., USA). The stock CP solution was prepared
under the sterile conditions by dissolving 100 mg of CP in 10 ml of sterile distilled
water, and the dosage of the drug was worked out on the basis of mg/kg body weight.
Nine parallel sets of experiments were carried out, where one of the three different
doses, i.e., 2 or 10 or 20 mg/kg body weight of CP was administered intraperitoneally
(i.p.), with the help of a sterile tuberculin syringe to the pregnant rats at either of
the three different, i.e., 12th or 15th or 18th day of gestation. These experiments
were designed according to the dose/gestation as: 2/12, 10/12, 20/12; 2/15, 10/15,
20/15; and 2/18, 10/18, 20/18. The pregnancy in all these experimental groups was
allowed to continue till delivery, and the tissues such as testes, liver and bone mar-
row collected from 1-day-old male pups for cytogenetic studies. In another set of
experiments, single dose of 20 mg/kg body weight of cytophosphamide was given at
12th day of gestation after dissecting the uteri horn for cytogenetic studies. Similar
procedure was adopted for collection of fetuses on 22nd day in the experiments with
delayed delivery. The control animals were divided into two major groups: one which
received the normal saline at three different gestations (12, 15, 18) parallel to the
experimental groups and the other with no injection at all. Because of the absence of
a significant difference between the two types of controls, the data was pooled and
the average values are used for comparison in the study.
Metaphase arrest The colchicines, a mitotic inhibitor (8 mg/kg body weight), were
injected to 1-day-old male pups, and pups sacrificed by cervical dislocation after two
hours of exposure.
2.1 Experimental Procedure 9
Chromosome preparation The fresh tissues from 1-day-old male pups and embry-
onic/fetuses were collected in a small glass petri dish or watch glass and minced with
a pair of scissors where required. This tissue suspension in saline was transferred to
the graduated conical centrifuge of the clumped cells and centrifuged at 800 rpm for
10 min and decanted.
Hypotonic treatment The pellet was suspended in prewarmed freshly prepared
0.56% potassium chloride solution or 0.9% sodium citrate solution in case of testic-
ular suspension and kept at 37 °C for 10–20 min. The cells were dispersed by gentle
pipetting and centrifuged at 800 rpm for 10 min.
Fixation The pellet thus obtained was fixed in 3:1 methanol: acetic acid. The fixative
was added slowly and changed thrice before the slide was prepared.
Slide preparation The layer of dispersed cell suspension was drawn out, and three
to four drops were added onto a precooled clean slide. The slide was then held close
to the flame for few moments, for the fixative to burn completely, leaving the slide
dry.
Chromosome staining and Giemsa banding The slide was stained with 5%
Giemsa stain or used for G-banding. The G-banding was done by the method of
Worton and Duff (1979), which is a modified technique of Seabright. The banding
was done at room temperature, and slides are rinsed in 0.15 M NaCl and exposed to
0.15 M NaCl containing trypsin solution for 30–120 s. The slides were again rinsed
with 0.15 M NaCl solution and then with 5% Giemsa for 5–10 min, followed by a
wash in distilled water and allowed to dry. The slides were observed for cytogenetic
features and photographed. The normal karyotype with 42 autosomes and X and Y
sex chromosomes of male Charles Foster rats as shown in Fig. 2.1.
The chromosomal features such as breaks, gaps, dicentrics, acentric fragments and
ring chromosomes were scored in unbanded metaphase plates as many times breaks in
chromosomes were not discernible clearly in banded metaphases. The chromosome
features observed in the study were divided into two groups: one including breaks,
gaps, acentric fragments, dicentrics and ring chromosomes (Fig. 2.2) and the other
including centromere spreading, chromatin bodies (Fig. 2.3) and aneuploidy. The
breaks and gaps in the former group were distinguished, and dicentric chromosomes
could not be confirmed by C-banding because most of the scoring of the features in
this group was done in unbanded metaphase plates. This was due to the reason of
missing breaks and gaps in swollen banded plates at times. The identification of such
features of centromeric spreading includes in the second group. The name ‘chromatin
bodies’ in this study are used as per convenience for fragmented chromosomes,
where fragmented pieces round up in different sizes to form chromatin bodies. The
probable mechanism of formation of such bodies and the end result is shown in
Fig. 2.3A, B. This feature seems apparently to be different from pulverization of
chromosomes. Further, the scoring of hyperdiploids (without exact multiples of basic
diploid number) was done as aneuploidy. Hypodiploid metaphase plates were only
10 2 Effect of Cyclophosphamide on Chromosomes
Fig. 2.1 Normal karyotype of male Charles Foster rats
Fig. 2.2 A–E Partial
metaphase showing breaks,
gaps, acentric fragments,
dicentrics and ring
chromosomes
2.1 Experimental Procedure 11
Fig. 2.3 A–D Partial
metaphase showing
centromere spreading (C and
D) formation of chromatin
bodies after extreme
fragmentation of
chromosome (A and B)
considered if consistently observed with the same chromosome number. The dose
and days relationship between nine parallel sets of experiments (already described)
was observed using arcsine transformation for proportions and a two-way analysis of
variance (F-values) in all the three tissues, i.e., testes, liver and bone marrow. Further,
chi-square test was applied to compare the differences between treated and control
experiments with respect to different chromosomal abnormalities. The correlation
coefficient ‘r’ was measured to examine the relationship between the three different
doses used and the aneuploidy in different chromosome groups. The correlation of
frequency distribution of aneuploidy of each chromosome between two different
groups was also measured and showing significant relationship.
A variety of abnormal chromosomal features were observed in testicular tis-
sue, liver and bone marrow of 1-day-old pups, obtained from pregnant rats, where
cyclophosphamide (CP) was administered in a signal dose of 2 or 10 or 20 mg/kg
body weight either at 12th or 15th or 18th day of gestation. The abnormal features
such as breaks, gaps, acentric fragments, dicentrics, ring chromatin, centromeric
spreading, chromatin bodies and aneuploids were observed in three different organs,
i.e., in tests, liver and bone marrow in the nine experiments designated according to
dose/day (gestation) as 2/12, 10/12, 20/12, 2/15, 10/15, 20/15 and 2/18, 10/18, 20/18
as well as in their respective controls.
The apartment observations of the frequency of total abnormal chromosomal fea-
tures both in testicular tissue and liver from 1-day-old pups showed a dose-dependent
increase irrespective of the gestational age at which they were exposed antenatally to
