Protective effect of green tea extract against cytotoxicity induced by enrofloxacin in rat

Enrofloxacin is bactericidal and has excellent activity against both Grampositive and Gram-negative pathogens.120 male and female adult albino rats (Rattus norvegicus) were allotted among three groups. The animal were given daily 75mg/kg of enrofloxacin intraperitonealy followed by injection of green tea extract 1%,1.5% and 3% for ten days. Various Structural and numerical chromosomal aberrations in bone marrow cells; mitotic activity and sperm head abnormality were recorded, quantitated, and statistically analyzed. Also DNA extraction and apoptosis detection in liver, kidney, and spleen was done; in addition to RNA damage was detected in lysate tissues. The intensity of apoptotic bands located at 200 b p; 400b p ; 600 b p; 800 b p and intact DNA measured by software Gel Pro program as maximum optical density values. Enrofloxacin had adverse effect on chromosomal and sperm head structure, also it induce apoptosis, necrosis and decrease total RNA in rat tissues. Green tea extract attenuate the enrofloxacin-related toxic effects. The most potent dose of Green tea extract was1% and the less effective was 3%.


INTRODUCTION
Enrofloxacin (ENRO) is a third generation fluoroquinolone antibiotic marketed specifically for use in veterinary medicine, inhibits DNA synthesis and is rapidly bactericidal against a broad spectrum of aerobic and some facultative anaerobic bacteria, including strains resistant to many other antimicrobial agents (Elmas et al., 2001).Enrofloxacin is eliminated by both renal and hepatic mechanisms, as well as in breast milk (Egerbacher et al., 2000).
The mode of action of enrofloxacin is interfering with bacterial DNA metabolism by the inhibition of two enzymes, Topoisomerase II (DNA gyrase) and Topoisomerase IV (Hooper and Wolfson, 1993).
Cytogenetic studies on enrofloxacin indicated that it was induce chromosomal aberration in cultures of human peripheral lymphocytes (Gorla et al., 1999).Also it inhibits cell proliferation and induced morphological changes in tendon cells (Yoon et al., 2004); in addition to it causes DNA damage in rat astrocytes Gurbay et al. (2006).Aral et al. (2008) postulated that enrofloxacin cause deterioration of motility and content of sperms as well as morphological abnormalities while Al-Nazawi (2008) suggested that enrofloxacin was safe on sperm traits for 10 days in male chicken.
Green tea is a type of tea made solely with the leaves of Camellia sinensis originates from China and has become associated with many other cultures in the Middle East.The regular consumption of green tea has long held value among traditional medical practices of promoting overall health (Sasazuki et al., 2004).
In green tea, the original polyphenols belong to the catechin family.Epigallocatechin gallate (EGCg) is considered to be the most active component in green tea, it constitute about 30% of the weight of dry leaves (Weisburger, 1997).
Tea catechins have garnered considerable attention as a result of beneficial effects on health, such as their observed antioxidant activity (Yoshino et al., 1994).Furthermore, the EGCg and other related catechins are thought to be responsible for several health benefits associated with the consumption of green tea, such as antimutagenic properties and anti-inflammatory activity (Nagle et al., 2006 andZaveri, 2006).
Green tea decrease chromosome aberrations (CA) in bone marrow cells (Ito et al., 1989 andEkram &abdella, 2005).Also green tea reduces DNA damage Glei and Pool-Zobel (2006) and protects the testicular function, in addition it act against sperm morphology changes Kang et al. (2000).
The aim of the present work is to investigate the side effect of enrofloxacin on chromosomes and sperms head morphology by cytogenetic methods.Also, detection of its side effect on DNA & RNA in different organs (liver, spleen, and kidney) by method of molecular biology and the protective role of green tea against enrofloxacin effects.

MATERIAL AND METHODS
In this study 120 albino rats, Rattus norvegicus (80 male & 40 female), varying from 100-120 gm in weight and aged from 10-12 weeks old were used.These rats were obtained from the National Research Center in Dukki, Cairo (N.R.C).
Enrofloxacin was injected intraperitonealy at dose level 75mg/kg calculated according to Paget and Barnes (1964) daily for ten days.Green tea extract was prepared according to (El beshbishy, 2005;Mohamadin et al., 2005 andkhan andkour, 2007).The solution of green tea extract was substituted in the place of water as the sole source of drinking fluid daily for ten days .Three doses from green tea extract were used 1%, 1.5% and 3% (10g, 15g and 30g of green tea in 1 L of boiling distilled water respectively).
The animals divided into three groups, group A&B contain 40 male and 40 female rats respectively used to study a chromosomal abnormalities and sperm head morphology by classical methods of cytogenetics, while group C contain 40 male rats used to molecular studies.Each group divided into 8 sub group, 5 rats were allotted to each sub group.
Metaphase spreads were prepared according to Yosida and Amano (1965).Fifty well metaphase spreads were examined / each animal.The type and frequency of chromosomal aberrations were recorded and photographed.
Protective effect of green tea extract against cytotoxicity induced by enrofloxacin in rat 47 Mitotic activity of the cells was calculated as the number of dividing cells including prophase and metaphase per 1000 cells.Cells with stickiness were considered as dividing cells.
Smears for sperm morphology were prepared and stained with eosin according to (Mukherjee et al., 1988).onethousand sperms were counted for each animal, and the abnormal shape involving the head was recorded.
Statistical analysis was carried out using the student (t) test (Snedecor, 1946).DNA extraction and apoptosis detection in tissues (liver, spleen, kidney) was done according to" salting out extraction method of (Aljanabi and Martinez, 1997) and modification introduced by (Hassab El-Nabi, 2004).
RNA damage was detected in lysate tissue (Hassab EL-Nabi et al., 2002).Gel was prepared using 1.8% electrophoretic grade agarose.All the gels of DNA and RNA were photographed with digital camera while the DNA and RNA were visualized at a312 nm UV light under a transilluminator.
Apoptotic bands appeared and located at 200 b p; 400b p; 600 b p and 800 b p. the intensity of apoptotic bands and intact DNA could be measured by software Gel pro program as maximum optical density values.

RESULTS
Various chromosomal aberrations are observed in the bone marrow cells of male and female rats treated with enrofloxacin and protected with different dose of green tea (1%, 1.5% & 3%).Both Structural and numerical types of aberrations and chromosomal stickiness are elucidated in group (A) (males) and group (B) (females).
(2) showed the average of chromosomal abnormalities of bone marrow cells of male rats treated with enrofloxacin and protected with green tea.It represented a very high significant increase in treated subgroup with enrofloxacin (132±8.952)than control subgroup (9.6±2.709), on the other hand it indicated that green tea made a high protective role against enrofloxacin especially green tea (1%)(17 ±3.438).(3) showed the average of chromosomal abnormalities of bone marrow cells of female rats treated with enrofloxacin and protected with green tea.It is showed a very high significant increase in treated subgroup with enrofloxacin (163±10.842)than control subgroup (12.6±3.062), on the other hand it was clear that green tea made a high protective role against enrofloxacin especially at concentration of (1%) (22 ± 3.322).(Doses 1%, 1.5 %, 3%) b Highly Significant (p<=0.01)T = Enrofloxacin (Dose 75mg/kg) c Very Highly Significant (p<=0.001)Tables (2&4) fig.(4&5) showed that the mean values of mitotic index decreased significantly after treated with enrofloxacin in males and females rats.But green tea extract especially at concentration (1%) repair a decrease in mitotic index caused by enrofloxacin.6) represented the incidence of abnormality in the shape of sperms per 1000 for each rats treated with enrofloxacin and protected with green tea, which include hammer shape (fig.7a), banana shape (fig.7b) , lack of hook (fig.7c) and amorphous (fig.7d).Enrofloxacin subgroup showed a very high significance increase in the average of total abnormality (134.6±4.674)compared with control subgroup (14.8±1.068).The highest incidence of abnormality in the shape of sperms of rats treated with enrofloxacin was amorphous while hammer shape was the lowest frequency.Green tea made a high protective role against sperm head abnormalities induced by enrofloxacin especially at concentration (1%) and (1.5 %).
Damage and optical density of DNA in rats treated with enrofloxacin and protected with green tea observed in liver (Fig. 8&9 and table 6), kidney (Figs 10&11 and table 7) and spleen (Figs 12&13 and tables 8) it represents that: Damage in DNA of rats treated with enrofloxacin increased when was compared with control so the optical density of apoptotic bands of DNA at 200; 400; 600 and 800 bp showed a very significant increase than control.On the other hand intact DNA decreased sharply than control.The obtained results showed that green tea reduce DNA damage induced by enrofloxacin especially at concentration (1%) and (1.5%).  ) showed electrophoretic patterns of RNA of rats at liver; kidney and spleen, respectively.It elucidated that damage in RNA of rats in treated subgroups increased in compared with control one so the total RNA decreased in enrofloxacin group than control one.On the other hand green tea made a high protective role against enrofloxacin as it acts against decreasing of RNA by enrofloxacin especially at concentration (1%) and (1.5%).

DISCUSSION
In the present work; fluoroquinolones antibiotic was targeted because it plays a major role in treating various diseases.This runs in accordance with (Hopkins et al., 2005) who stated that fluoroquinolones such as enrofloxacin are broad spectrum antimicrobials highly effective for treatment of variety of clinical and veterinary infections.
This study presented very important plants such as green tea.Green tea aqueous extract have found to inhibit the incidence of enrofloxacin induced genotoxicity in rat's liver, kidney, spleen, bone marrow and sperm, suggesting its potential as antigenotoxic agents.This is in agreement with (El-Ghonaimy, 2005) who found green tea plays high protective action as it is antimutagenic agent.
In the present study it has been used different doses from green tea (1%, 1.5% and 3%) to detect the most effective dose against enrofloxacin infections.Similarly, Mohamaden et al. (2005) used different concentrations of green tea extract GTE (0.5, 1.0 and 1.5%) as sole source of drinking water in rats to attenuate cyclosporine A (CsA)-induced renal dysfunction, and found that GTE (1%) was the best dose.
In the present study it has been observed that the number of deletion was a very highly significantly increased at all treated groups with enrofloxacin compared to that of the control one in both males and females.This in accordance with (Khater, 1991) who found that deletions were statistically significant from that of control group in rats treated with a mutagenic agent.
A very high significant increase was obtained in the percentage of fragments of all treated groups with enrofloxacin in both males and females compared to that of control.This result is in accordance with (Abo-Ghanema, 2009) who found that ciprofloxacin induce significant chromosomal aberrations, especially fragments in rats.
It is obvious from the present study that the statistical analysis of chromosomal ring, centromeric attenuation, centric fusion and end to end association, of chromosomes of bone marrow cells of rats treated with enrofloxacin showed a very highly significant increased in the value than that of control in both males and females.These results were disagreement with (Gorla et al., 1999) who found that after treatment with ciprofloxacin and enrofloxacin no significant differences in these types of chromosomal aberrations of human lymphocytes were observed.
Significantly increased of gap was observed in this study in treated animals with enrofloxacin than control and this was due to the local loss of both DNA and chromosomal basic protein, this loss occur on a chromatid in the locus and doesn't represent real discontinuities in the chromosome (Stoian and Raicu, 1975).It was occurred as a result of primary lesions which disrepair to give aberrations (Evan, 1977).
This study cleared, enrofloxacin induce gap and break aberrations so they increase significantly in treated groups than control and that is agreement with (Gorla et al., 1999) who found that gap and break were increase significantly in treated lymphocytes of human with enrofloxacin.
Aneuploidy in general arise as a non disjunction of homologous chromosomes at meiosis, or by non disjunction of sister chromatids at mitosis.The failure of disjoin or separate accurately can occur at any nuclear division in which the event occurs at the time occurrence.Non disjunction at meiosis gives rise to gametes with one more or less chromosome than usual.If such gametes are viable and fuse to produce a zygote, the zygote will be trisomic or monosomic for non disjunction chromosomes (Avers, 1980).
In the present study the numerical aberrations showed a significant difference in all treated subgroups with enrofloxacin than control.
The value of chromosome stickiness increased in bone marrow cells of rats treated with enrofloxacin in both males and females than control.Stickiness is due to the process of depolymerization of DNA, thus making the chromosome surface becoming sticky.Stickiness has been regarded later as physiological and unspecific disturbance attributed to the action of proteins on chromosome or form improper folding of the chromosome fibers into chromatids and thus chromosome become attached to each other by means of sub chromatid bridges (Brogger, 1974).
The mean values of mitotic index decreased in males and females rats after treated with enrofloxacin.Mitotic index is a measure for the proliferation status of a cell population.The apparent lower values for mitotic index reported in the present study may be due to decrease in cell number arrested in metaphase (Wissmuller, 1971).
In the present study green tea acts as highly protective agent so aberrations decreased in all protected subgroups than treated one with enrofloxacin and that agreement with (Ito et al., 1989) who arrive to results that green tea is highly protective agent against aflatoxin B due to its antimutagenic activity.
Also, the present study showed that concentration of green tea extract (1%) and (1.5%) were the more protective doses against chromosomal aberrations induced by enrofloxacin than green tea (3%) in both male and female rats.
In the present study, the sperm head abnormalities as amorphous and banana like was the highest incidence of aberration in all groups, while lack of hook and hammer shape was the lowest frequency in all groups after treatment with enrofloxacin.Zowail (1992) recorded five types of sperm head abnormalities amorphous, banana like, lack of hook, two head and folded in rats treated with piroxicam.The same author stated that amorphous was the highest incidence of aberration in all groups.
This study showed that rats treated with enrofloxacin revealed an increase in the frequency of total epididymal sperm head abnormalities.These results in accordance with Aral et al. (2008) who suggested that enrofloxacin would leadb disruption of spermatogenesis in the testes causing deterioration of motility and content of sperms as well as morphological abnormalities.But these results were disagreement with Al-Nazawi (2008) who suggested that enrofloxacin was safe on sperm traits in male chicken for 10 days.
In an attempt to explain the different mechanisms involved in the induction of the abnormal morphology of the sperm heads, Kaczmarski (1972) stated that incomplete condensation of chromatin and the presence of large vacuoles and canals containing remnants of cytoplasm in various regions of the head is the cause of failure Protective effect of green tea extract against cytotoxicity induced by enrofloxacin in rat 53 of sperm to pass through the final steps of maturations occurring normally during spermatogenesis.Moreover, Topham (1980) mentioned the agents which accumulate in the testis can cause alterations in testicular DNA and disrupt the process of differentiation of spermatozoa directly.
It is clear from the obtained results that green tea made a high protective role against sperm head abnormalities induced by enrofloxacin especially at concentration (1%) and (1.5%),this trend was encouraged by Kang et al.(2000) who said that green tea protects the testicular function and act against sperm morphology changes in rats.
The results of the present study showed that enrofloxacin induced genotoxic effects including DNA damage (apoptosis and necrosis) in the treated rats.
Apoptosis is generally considered as an energy-dependent process that requires active participation of many proteins and other cellular macromolecules.Apoptosis seems to be induced by mild genotoxic stimuli; the strength of stimuli increases the cell death mode shifting it towards necrosis.This seems to be due to the fact that most the intense genotoxic stimuli damage the proteins or genes that make these proteins and other cellular macromolecules which may be required for apoptosis (Singh, 2000).
Apoptosis is mediated by members of the caspase family of proteases, and eventually causes the degradation of chromosomal DNA.
In this work green tea extract decreased the mutagenesis of enrofloxacin and the term antimutagen was originally used to describe an agent that reduces the apparent yield of spontaneous and induced mutations (Morita et al., 1978).(Kada, 1983) has classified the mechanisms of antimutagenesis into two major processes.One is desmutagenesis in which factors act directly on mutagens or their precursors and inactivate them.The other is bio-antimutagenesis, in which factors act on the processes of mutogenesis or repair DNA damages to result in a decrease in mutation or damage frequency.Antimutagens effective in stimultaneous treatment with mutagens may act as desmutagens which chemically or enzymtically inactivate of promutagens or inhibit the metabolic activation of promutagens (Kurada et al., 1992).
On the other hand it has been shown that polyphenols, including catechins, can act not only as antioxidants, but also pro-oxidants under certain conditions (Sakihama et al., 2002).Several invitro experiments have demonstrated that catechins can induce oxidative DNA damage, including the formation of 7 -hydro-8-oxo-2'-deoxyguanosine (8-oxodG) (Oikawa et al., 2003).
In the present work enrofloxacin decreased the total RNA in liver, spleen and kidney.This trend was encouraged by Yoon et al. (2004) who found that morphological changes and inhibition of cell proliferation induced by enrofloxacin on tendon cells are a result of decrease level of mRNA in cell.On the other hand, green tea extract made a high protective role against total RNA decreasing induced by enrofloxacin.

Fig. 2 :
Fig.2: Average of chromosomal abnormalities observed in bone marrow cells of male rats treated with enrofloxacin and protected with green tea.

Fig. 3 :
Fig. 3: Average of chromosomal abnormalities observed in bone marrow cells of female rats treated with enrofloxacin and protected with green tea.

Fig. 4 :
Fig.4: The mean value of mitotic index of bone marrow cells of male rats treated with enrofloxacin and protected with green tea.

Fig. 5 :Fig. 8 :
Fig. 5: The mean value of mitotic index of bone marrow cells of female rats treated with enrofloxacin and protected with green tea.

Table ( 1
): Average of chromosomal abnormalities observed in bone marrow cells of male rats treated with enrofloxacin and protected with green tea.

Table ( 3
): Average of chromosomal abnormalities observed in bone marrow cells of female rats treated with enrofloxacin and protected with green tea.

Table ( 2
): Numerical chromosomal abnormalities and mitotic index in bone marrow cells of male rats treated with enrofloxacin and protected with green tea.

Table ( 4
): Numerical chromosomal abnormalities and mitotic index in bone marrow cells of female rats treated with enrofloxacin and protected with green tea.

Table ( 5
): Incidence of the sperm head abnormality per thousand after treatment with enrofloxacin and protection with green tea.

Table ( 6
) Optical density of intact and apoptotic fragments of DNA at 200, 400, 600 and 800 bp in liver of rats treated with enrofloxacin and protected with green tea.

Table ( 7
) Optical density of intact and apoptotic fragments of DNA at 200, 400, 600 and 800 bp in kidney of rats treated with enrofloxacin and protected with green tea.

Table ( 8
) Optical density of intact and apoptotic fragments of DNA at 200, 400, 600 and 800 bp in spleen of rats treated with enrofloxacin and protected with green tea.