I. Introduction
Cytotoxic/immunosuppresive drugs are agents used to
treat and cure many forms of malignancies. Because of its potent
immunosuppressive properties, it has received considerable attention for the
control of several clinical settings where the goal of therapy is to suppress
an unwanted immune response (Paul and Bruce, 1991). The major current
indications for drugs include the control of organ rejection after
transplantation, prevention of Rh hemolytic disease of the new born and
non-neoplastic disorders associated with altered immune reactivity (Annward et
al, 1990; Paul and Bruce, 1991; Diasio and Lobuglio, 1991; Chaki, 1999). These
drugs work by targeting and damaging cells that grow at rapid rate as antibody
producing cells of the immune system, blood cells, hair cells, gonadal cells
and malignant cells (Diasio and Lobuglio, 1991). Inspite of its serious side
effects, they can be of great value in treatment, they can prolong life,
preserve function, reduce symptoms, and sometimes may serve to put the disease
into remission (Oka and Yoshimura, 1996).
AZA is immunomodulatory drug often used to treat
inflammatory bowel disease, autoimmune diseases, prevent rejection of
transplanted organs and also used as anticancer drug (Schein and Winokur, 1975;
Tage-Jensen et al, 1987; Dejaco et al, 2001; McMullaen et al, 2001; Langer et
al, 2003; Marcen et al, 2003). It is inhibitor of purine metabolism leading to
DNA damage. Upon its administration, it rapidly converted into several
compounds, including the active 6-mercaptopurine (Diasio and Lobuglio, 1991;
William et al, 1998). AZA can affect rapidly growing cells including bone
marrow and gastrointestinal cells, resulting in leukopenia, thrombocytopenia,
increased susceptibility to infections and hepatotoxicity (Arber et al, 1991;
Rosenkranz and Klopman, 1991; Olshan et al, 1994; Johnson et al, 1995;
Rojapakse et al, 2000; Lowry et al, 2001; Kersten et al, 2002; Norgad et al,
2004).
Treatment of certain types of cancer with cyclical
combined chemotherapy with or without radiotherapy has dramatically increased
the rate of long-term remission. The increased survival of patients has focused
attention on the chronic effects of these cytotoxic agents on the function of
normal tissues, which is not manifested until the damage is extensive (Annward
et al, 1990). Unfortunately, the destructive effects of these treatments on
spermatogenesis are well documented. Iatrogenic infertility and sterility are
serious side effects of cytotoxic chemotherapy in young patients with
relatively normal life expectation (Heikens et al, 1996; Gerres et al, 1998;
Soriano et al. 2000; Silva et al, 2002; Tal et al, 1985; Rueffer et al, 2001;
Das et al, 2002). AZA severely affect spermatogenesis in rat, it significantly
lowered sperm count in couda epididymis and caused dose dependent damage of the
seminiferous tubules (Iwasaki et al, 1996). Furthermore, patients received a
combination of Cyclosporine- Azathioprine and Prednisone has low testesterone
level and impairment of hypothalamic pituitary gonadal axis (Ramirez et al,
1991). The most serious complication among patients undergoing
immunosuppressive therapy is the risk of developing cancer. Many of these drugs
used have mutagenic properties and also contribute to increased cancer risk
(Schein and Winokur, 1975; Mitrou et al, 1979; Baker et al, 1987). AZA is
mutagenic, genotoxic and several types of tumors are associated with prolonged
treatment with it (Clark, 1975; van Went, 1979; Nagafuchi and Miyazaki, 1991;
Langer et al, 2003; Marcen et al, 2003).
Cytotoxic drugs disturb oxidant-antioxidant balance
and the oxidative damage to sperm, testis and genetic material is thought to be
responsible for serious effects on male fertility and genomic stability
(Ahotupa and Huhtaniemi, 1992; Michael et al, 1999; Blasiak et al, 2002). The
potential role of dietary antioxidants as tocopherol, ascorbic acid, b-carotin, etc to reduce
the activity of free radical-induced reactions has drawn increasing attention
(McCall and Balz, 1999; Oliveira and Fortes, 2003).
The purpose of this study
was to assess the effect of short and long term AZA treatment on seminiferous
tubules and bone marrow chromosomes and the possible protective effect of
vitamin C in adult male albino rats.
II.
Materials and methods
A. Drug
A commercial available
formulation of AZA tablets 50 mg was used.
B. Animals
80 adult male albino rats
(150-200 gm) body weight were used. The animals were kept in standard housing
conditions and freely supplied with food and water for one week before the
experiment.
C. Experimental
design
The animals were divided into
two major groups
i. Short term experiment which include:
Group 1: Received AZA (150
mg/kg body weight) gavaged as a single dose.
The animals sacrificed after
14 days post treatment.
The same dose was previously
used to evaluate hepatotoxicity and carcinogenecity of AZA in rat (IARC, 1981;
Arber et al, 1991). The used dose is larger than that of human as the smaller
the animal, the larger the dose/kg b.wt (Paget and Barnes, 1964)
Group 2: Received AZA as in
group 1 in conjunction with vitamin C (100 mg/kg body weight) gavaged and maintained
for 14 days after AZA treatment.
ii. Long term experiment
Group1: Received AZA (15mg/kg
body weight) gavaged daily for two months. The used dose is about 1/10 of the
acute dose used in this study.
Group 2: Received AZA as in
group1 simultaneously with vitamin C (100 mg/kg b.wt for two months
Two control groups 10 animals
each were used for each experiment:
Control 1: Received distilled
water orally
Control 2: Received vitamin C
(100mg/kg b.wt) dissolved in distilled water.
At the end of each experiment
the animals were sacrificed by decapitation after ether anesthesia and were
subjected to the following studies:
D.
Histological examination
Both testes were removed and
weighted then fixed quickly in BouinÕs fluid and processed for light microscopic
examination using haematoxylin and eosin (H & E) (Drury and Wallington
1980).
E. Cytogenetic analysis
Bone
marrow from one femur was obtained to perform analysis of chromosomal
aberrations and from other femur to analyze micronuclei.
1. Chromosomal aberrations
The
animals were sacrificed 1-2 hrs after injection of 4 mg/kg b.wt colchicine.
Bone marrow preparation was made according to Giri et al, 1986. The cells
spread into clean slides. The slide were air-dried stained with Gur Giemsa and
50 well spread metaphases per animal were selected for analysis of chromosomal
aberrations. The mitotic indices were calculated from 1000 cells per animals.
2. Micronucleus technique
Micronucleus
preparations were prepared according to Schmid et al, 1976. 1000 polychromatic
erythrocytes were demonstrated for each animal. The micronuclei represent
condensed or chromosome fragments that remain after the nucleus expelled.
F. Statistical analysis
Evaluation of mean frequencies between treated
and control groups by StudentÕs t-test. Results were considered statistically
significant at P<0.05.
III. Results
A. Histological examination
Both short and
long-term AZA treatments significantly lowered testicular weight compared with
the control (Table 1).
Administration of vitamin C for 14 days after single large dose significantly
increased testis weight compared with AZA treated groups. While
co-administration of vitamin C with AZA for two months significantly restore
testicular weight to normal (Table 1).
Microscopical examination
of testicular tissues from the control and vitamin C treated animals showed
normal cytoarchitecture and maturation of germinal epithelium (Figures 1A).
This in sharp
contrast to the testis of AZA treated animals (Figures 1B, Figures 2A). In these animals treated with 150 mg/kg
b.wt, there were loss of both the basic tubular morphology and most of germinal
epithelium. Relatively, few spermatogonial stem cells were observed, the cells
displayed hyperchromatic nuclei, and vaculated cytoplasm (Figures 1 B, C, D). Testicular tissues of some animals showed
irregular and severely regressed tubules with predominant vacuolated sertoli
cells and widening of interstitial spaces due to presence of odema (Figures 1 E, F). Administration of
vitamin C 14 days post AZA treatment caused a remarkable sparing of germ cell
line. All types of germinal cells were present within the epithelium of many
tubules. Tubular architecture was preserved and germinal epithelium showed a
normal maturation progression (Figures 1
G, H). Testicular tissue of the rats received AZA (15 mg/kg b.wt) revealed
moderate affection of many seminiferous tubules. Large number of germ cells
were detached from sertoli cells and sloughed in the lumen of the tubules
leading to obstruction and enlargement of the testis
IV. Discussion
Cytotoxic
drugs that are widely used as immunosuppressive and anti-inflammatory agents in
patients with neoplastic conditions are of long-range concern due to the
problem of cumulative organ toxicity that is not manifested until damage is
extensive. These considerations have arisen because of their wide spread use in
recent years (Oka and Yoshimura, 1996; Bunn and Kelly, 1998). In view of the
marked cytotoxicity of most anticancer drugs, it exerts adverse effects in
young patients (Whitehead et al, 1981). This study has provided an insight into
some fertility problems and genotoxicity associated with AZA treatment.
Testicular weights and microscopic examination of testicular tissue showed that
short and long term administration of AZA have diverse effects on male
fertility. Single large dose of AZA (150 mg/kg b.wt) showed germinal aplasia
and the seminiferous tubules were extremely atrophied, most of the cells within
the tubules were sertoli cells and occasionally germ cell with pyknotic nuclei
and vaculated cytoplasm were seen. There is absence of many stages of
spermatogenesis compared with the control group. The reduction in the testis
weight is indirectly indicative of the effect on spermatogenesis. Dhabhar et
al, 1993, proved that Sertoli cells which secrete inhibin are resistant to
these cytotoxic agents. Ramirez et al, 1991; Iwasaki et al, 1996, proved that
AZA induced impairment of spermatogenesis by direct inhibition of germinal
epithelium or indirect by influencing the axis between hypothalamus-pituitary
and gonads. These effects were proved morphologically by testicular atrophy and
azoospermia. Furthermore, Annward et al, 1990, found that rapidly dividing
cells are more sensitive to cytotoxic drugs than quiescent cells, hence
sterility and ovarian dysfunction appear to be less common in females treated
with these drugs than in males indicating that the ovary with its lower germ
cells proliferative rate may be partially protected from cytotoxic drugs
(Annward et al, 1990). Furthermore, Mclachlan et al, 1996, observed that acute
withdrawal of testesterone by the use of leydig cell cytotoxin produce
destructive pattern of spermatogenic cells degeneration with sharp increase in
the number of pyknotic nuclei and vaculated cytoplasm which was observed in our
study.
Using similar criteria, Dekretser et al,
1972, reported that isolated germinal epithelium damage is rare and that leydig
cell function is nearly always impaired as well, and other reports described
gynecomastia in pubertal boys treated with cytotoxic drugs that was
manifestation of leydig cell dysfunction (Sherin et al, 1978). These mean that
low testestorone level due to damage of leydig cells responsible for the
morphological changes observed in the testis. On the other hand testicular
tissue of animals treated with AZA (15 mg/kg b.wt) orally daily for two months
revealed moderate degenerative changes in most seminiferous tubules. Many germ
cells were detached from Sertoli cells and sloughed in the lumen of
seminiferous tubules, the slaughed cells contained desqumated spermatid and
spermatocytes. In the present study, adhesion of round spermatids approved to
be lost resulting in their sloughing into the lumen. Richburg and Boekelheide,
1996, proved that chronic reduction of testicular testesterone level in
testesterone suppressed rat, reduce the number of spermatogonia and
spermatocytes to 60% of normal suggesting the role of testesterone in the
maintenances of these cell population. Also absence of testesterone lead to
loss of spermatid adhesion, preventing their further maturation. Aumuller et
al, 1992; lee et al, 1999, proved the basis of testesterone dependency of
spermatid/ Sertoli cell cytoskeleton. Normally junctional area termed the
ectoplasmic specialization develops between Sertoli cells and round spermatids,
disrupted in the absence of testesterone and caused sloughing of spermatids
into the lumen.
The most popular mechanism of AZA induced
cellular damage was lipid peroxidation. AZA with other cytotoxic drugs are
associated with the induction of oxidative stress by generation of free
radicals and reactive oxygen species (ROS), which interfere with testicular
gametogenic activities. Our results showed that vitamin C provide significant
protection of testicular tissue and spermatogenesis when administered in both
AZA treatments. This is in agreement of Das et al, 2002, who proved testicular
protection against cyclophosphamide toxicity by vitamin C administration, this
suggesting the role of vitamins in prevention of cytotoxic drug-induced
testicular damage.
The sperm are extremely sensitive to free
radicals damage due to active generation of free radicals, lack of defensive
enzymes and high concentration of polyunsaturated fatty acids. Without proper
membrane fluidity, enzymes are activated which can lead to impaired motility,
abnormal structure, loss of viability and death of sperms (Baker et al, 1996;
Hsu et al, 1998). These factors make the health of sperms critically dependent
upon antioxidant. Michael et al, (1999), demonstrated that free radicals or
oxidative damage to sperm is thought to be responsible for many cases of
idiopathic oligospermia with high levels of free radicals found in semen of
infertile men. Fraga et al, 1991; Chen et al, 2001, observed that when dietary
vitamin C was reduced the seminal ascorbic acid decreased and the number of
sperm with damaged DNA increased. These results indicated that dietary vitamin
C plays a critical role in protecting against sperm damage.
In our study both small and large doses of
AZA increased number of micronucleus, structural and numerical chromosomal
aberrations (with large dose only). van Went, 1979, observed a dose-dependent
increase in the number of the cells with micronucleus in rat and mice caused by
AZA treatment and increased number of structural chromosomal aberrations in
lymphocyte cultures of children on long-term AZA therapy. Both treatments with
AZA cause a significant reduction in the number of dividing cells. Nagafuchi
and Miyazaki, 1991, observed a dose dependent increase in DNA single strand
breaks with concomitant cytotoxicity associated with AZA treatment.
The role of vitamin C in reducing
genotoxicity induced by many agents has been proved (Gajecka et al, 1999;
Nefic, 2001; Siddique et al, 2005). Vitamin C reduced the clastogenic effect
induced by anticancer drugs dexorubicine and idarubicin (Antunes and Takahashi,
1998; Tavares et al, 1998; Pillanse et al, 2002; Blasiak et al, 2002). However,
in our results vitamin C did not exert any protective effect on AZA induced
structural chromosomal aberrations. Pillanse et al, 1990, found that ascorbic
acid provide protection from cyclophosphamide induced teratogenic effect in
mouse embryo, this protection is not associated with prevention of DNA strand
breaks. Furthermore, vitamin C did not provide protection from genotoxic effect
of toxophene, dichorovos and nitrosomorpholine compounds (Cabrera 2000;
Robichova et al, 2004).
Large dose of AZA associated with
increased polyploidy cells. Motwani et al, 2000, suggested that cell with
compremized G1 checkpoint in response to microtubule inhibitors enter S phase
with 4n DNA, endoreduplicate and become polyploid cells. We suggested that
large dose of AZA may act as microtubules inhibitors. Ferguson et al, 1996,
observed increased number of polyploidy cells associated with high dose of
amsacrine (antileukemic drug). Meanwhile, the use of small and large dose of
this drug led to chromosomal fragments. Treatment of the rats with vitamin C 14
days following AZA treatment (150 mg/kg. b.wt.) caused a significant reduction
in polyploidy cells. Motwani et al, 2000, showed that endoreduplication and
polyploidation can prevented by inhibition of cycline-dependent kinase,
resulted in the arrest of cells in pseudo G1 state and dramatic decrease in
cells containing >4n DNA. Thomas et al, 2005, proved that vitamin C delay
the accumulation and activation of cell cycle control kinases.
Defects
in cell cycle checkpoints can lead to chromosome abnormality, aneuploidy, and
genomic instability, all of which can contribute to tumorigenesis (Lannutti et
al, 2005; Vries et al, 2005). The antitumour effect of vitamin C has been
proved (Roomi et al, 2005; Thomas et al, 2005). They proved that vitamin C
transiently arrest cancer cell cycle progression in S phase and G (2)/M
boundary by delaying the accumulation and activation of cell division control
kinases/cycline complex. We suggesting that vitamin C provide genomic stability
by preventing polyploidy
In conclusion effort should
be made to identify more careful design of non-toxic chemotherapy regimes.
Vitamin C provided significant protection to the spermatogenic cells and
provided genomic stability but did not protect the cells from structural
chromosomal aberration. It may be used with other antioxidants for full
protection of genetic material.
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