CBI 7303
No. of Pages 10, Model 5G
11 March 2015 Chemico-Biological Interactions xxx (2015) xxx–xxx 1
Contents lists available at ScienceDirect
Chemico-Biological Interactions journal homepage: www.elsevier.com/locate/chembioint 5 6
4
Evaluation of the toxic potential of cefotaxime in the third instar larvae of transgenic Drosophila melanogaster
7
Rahul Sachdev, Smita Jyoti, Falaq Naz, Yasir Hasan Siddique ⇑
8
Drosophila Transgenic Laboratory, Section of Genetics, Department of Zoology, Faculty of Life Sciences, Aligarh Muslim University, Aligarh, Uttar Pradesh 202002, India
3
10 9 11 1 2 3 6 14 15 16 17 18 19 20 21 22 23 24 25
a r t i c l e
i n f o
Article history: Received 23 July 2014 Received in revised form 25 December 2014 Accepted 3 March 2015 Available online xxxx Keywords: Tissue damage Oxidative stress Comet assay b-Galactosidase Drosophila melanogaster (hsp70-lacZ)Bg9
a b s t r a c t The present study was carried out to evaluate the toxic potential of cefotaxime in the third instar larvae of transgenic Drosophila melanogaster (hsp70-lacZ)Bg9. Cefotaxime at final concentration of 10, 20, 40, 60 and 80 lg/ml was mixed in the diet and the larvae were exposed to the selected doses for 6, 12, 24, 48 h. The hsp70 expression, trypan blue exclusion test, in situ histochemical b-galactosidase activity, lipid peroxidation, total protein content, glutathione (GSH) content, glutathione-S-transferase (GST) activity, protein carbonyl content, caspase 3 and 9 activity, apoptotic index and comet assay were taken as parameters for the study. The larvae exposed to 40, 60 and 80 lg/ml for 12, 24 and 48 h showed a dose and duration dependent significant increase in the activity of b-galactosidase and lipid peroxidation but decrease in the total GSH content as compared to unexposed larvae. The decrease in protein content was observed in the larvae exposed to 40, 60 and 80 lg/ml of cefotaxime for 24 and 48 h. The larvae exposed to 40, 60 and 80 lg/ml of cefotaxime for 24 and 48 h showed a dose and duration dependent increase in the tissue damage, GST, caspase 3 and 9 activity, PC content, apoptosis and the DNA tail length (comet assay). The result suggests that the cefotaxime is toxic at 40, 60 and 80 lg/ml of doses for the third instar larvae of transgenic D. melanogaster (hsp70-lacZ)Bg9. Cefotaxime at 10 and 20 lg/ml was not toxic for any duration of exposure. Ó 2015 Published by Elsevier Ireland Ltd.
27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44
45 46
1. Introduction
47
Cefotaxime (CTX), a third generation broad spectrum cephalosporin is commonly used as perioperative anti-microbial prophylactic in orthotropic liver transplantation (OLT) [1]. It provides protection against wide range of bacteria [2]. It has been reported for the adverse effects in humans showing an increase in serum aspartate amino transferase level and the occurrence of pseudomembranous colitis [3]. It has been reported for extremely low toxicity profile with an intravenous LD50 values in the range of 9–10 g/kg [4]. The higher doses of cefotaxime (600 mg/kg/day) showed a significant mortality in neonatal and adult mice [5]. Antibiotics have been used in therapeutics for more than 70 years, though no rational standardized approaches have been defined for the treatment in humans [6]. Due to ethical reasons, the toxic evaluations cannot be performed directly on humans. The fruit fly Drosophila melanogaster has been extensively used in basic and applied research for various toxicological evaluations and human disorders [7–11]. Fly has advantage over mammalian model because of the ease of laboratory maintenance and genetic
48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64
⇑ Corresponding author. E-mail address: [email protected] (Y.H. Siddique).
manipulations. As an alternative to higher animal, Drosophila has been well documented and recommended for toxicological evaluations. About 50% of the fly protein sequences have mammalian homologs and the results obtained can be extrapolated by using various mathematical models [12]. Heat shock proteins (HSPs) were initially reported to be expressed in response to heat but their expression is also triggered in response to diverse range of stress [13]. In the recent years, hsp70 has been considered to be one of the candidate genes for predicting cytotoxicity against environmental chemicals [14]. The present study was conducted to evaluate the toxic potential of cefotaxime using hsp70 expression, tissue damage, X-gal staining, lipid peroxidation, protein content, apoptosis and comet assay as parameters in the third instar larvae of transgenic D. melanogaster (hsp70-lacZ)Bg9 at various doses and durations of exposure.
65
2. Materials and methods
80
2.1. Fly strain
81
A transgenic D. melanogaster line expressing bacterial b-galactosidase in response to stress was used in the present study [15].
82
http://dx.doi.org/10.1016/j.cbi.2015.03.004 0009-2797/Ó 2015 Published by Elsevier Ireland Ltd.
Please cite this article in press as: R. Sachdev et al., Evaluation of the toxic potential of cefotaxime in the third instar larvae of transgenic Drosophila melanogaster, Chemico-Biological Interactions (2015), http://dx.doi.org/10.1016/j.cbi.2015.03.004
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88
In the afore mentioned strain, the transformation vector is inserted with a P-element, i.e., the line contains wild type hsp70 sequence up to lacZ fusion point. The flies and larvae were cultured on standard Drosophila food containing agar, corn meal, sugar and yeast at 24 ± 1 °C [16,17].
89
2.2. Experimental design
84 85 86 87
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The final concentrations of cefotaxime, i.e., 10, 20, 40, 60 and 80 lg/ml were established in diet. The third instar larvae were allowed to feed for 6, 12, 24 and 48 h.
93
2.3. Soluble O-nitrophenyl-b-D-galactopyranoside (ONPG assay)
94
The expression of hsp70 was quantified by performing soluble O-nitrophenyl-b-D-galactopyranoside (ONPG) assay as described by Nazir et al. [16]. After giving a wash of phosphate buffer, the larvae were placed in microcentrifuge tubes (20 larvae/tube; 5 replicates/group), permeabilized for 10 min by acetone and incubated overnight at 37 °C in 600 ll of ONPG buffer. After incubation for the desired duration the reaction was stopped by adding 300 ll of Na2CO3 and the extent of the reaction was quantified by measuring absorbance at 420 nm [18,19].
90 91
95 96 97 98 99 100 101 102
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2.4. In situ histochemical b-galactosidase activity
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The larvae (10 larvae/treatment; 5 replicates/group) were dissected out in Pole’s salt solution (PSS) and X-gal staining was performed using the method as described by Chowdhuri et al. [18]. The larvae explants were fixed in 2.5% glutaraldehyde, washed in 50 mM sodium phosphate buffer (pH 8.0) and stained overnight in X-gal staining solution at 37 °C in the dark.
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2.5. Trypan blue exclusion test
111
For studying the extent of tissue damage in larvae caused by the exposure to different dosages of cefotaxime dye exclusion test was performed [16,20]. Briefly, the internal tissues of larvae were explanted in a drop of PSS, washed in phosphate buffer saline (PBS), stained in trypan blue (0.2 mg/ml in PBS) for 30 min, washed thoroughly in PBS, and scored immediately for dark blue staining. About 50 larvae per treatment (10 larvae per dose; 5 replicates group) were scored for the trypan blue staining on an average composite index per larvae: no color = 0; any blue = 1; darkly stained = 2; large patches of darkly stained cells = 3; or complete staining of most cells in the tissue = 4 [20].
112 113 114 115 116 117 118 119 120 121
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2.6. Preparation of larval homogenate
123
The larvae (10 larvae/dose; 5 replicates/group) were homogenized in 1 ml of cold homogenizing buffer (0.1 M phosphate buffer containing 0.15 M KCl; pH 7.4). The supernatant after centrifugation at 9000g was used for estimating lipid peroxidation, total protein, glutathione content, glutathione-S-transferase activity and protein carbonyl content.
124 125 126 127 128
129 130 131
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2.6.1. Lipid peroxidation assay Lipid peroxidation assay was performed as described earlier using 1,1,3,3-tetramethoxy propane as a standard [21,22]. 2.6.2. Protein estimation The protein content in all the treated as well as untreated groups were estimated according to the method of Bradford (1976) using bovine serum albumin (BSA) as a standard [23].
2.6.3. Estimation of glutathione (GSH) content The glutathione (GSH) content was estimated colorimetrically using Ellman’s reagent (DTNB) according to the procedure described by [24]. The supernatant was precipitated with 4% sulphosalicylic acid (4%) in the ratio of 1:1. The samples were kept at 4 °C for 1 h and then subjected to centrifugation at 5000 rpm for 10 min at 4 °C. The assay mixture consisted of 550 ll of 0.1 M phosphate buffer, 100 ll of supernatant and 100 ll of DTNB. The OD was read at 412 nm and the results were expressed as l moles of GSH/gram tissue.
136
2.6.4. Estimation of glutathione-S-transferase (GST) activity The glutathione-S-transferase activity was determined by the method of Habig et al. [25]. The reaction mixture consist of 500 ll of 0.1 M phosphate buffer, 150 ll of 10 mM CDNB, 200 ll of 10 mM reduced glutathione and 50 ll of supernatant. The OD were take at 340 nm and the enzyme activity was expressed as l moles of CDNB conjugates/min/mg protein.
146
2.6.5. Estimation of protein carbonyl content The protein carbonyl content was estimated according to the protocol described by Hawkins et al. [26]. The larvae homogenate was diluted to a protein concentration of approx. 1 mg/ml. About 250 ll of each diluted homogenate was taken in Eppendorf centrifuge tubes separately. To it 250 ll of 10 mM 2,4-dinitrophenyl hydrazine (dissolved in 2.5 M HCl) was added, vortexed and kept in dark for 20 min. About 125 ll of 50% (w/v) trichloroacetic acid (TCA) was added, mixed thoroughly and incubated at 20 °C for 15 min. The tubes were then centrifuged at 4 °C for 10 min at 9000 rpm. The supernatant was discarded and the pellet obtained was washed twice by ice cold ethanol:ethyl acetate (1:1). Finally the pellets were re-dissolved in 1 ml of 6 M guanidine hydrochloride and the absorbance was read at 370 nm.
153
2.7. Assay for caspase-3 (Drice) and caspase-9 (Dronc) activities
167
For estimating the activities of caspase 3 and 9 the midguts of the larvae were collected in PBS. The assay was performed according to the manufacturer protocol with some modification (Bio-Vision, CA, USA). The assay was based on spectrophotometric detection of the chromophore p-nitroanilide (pNA) obtained after specific action of caspase-3 and caspase-9 on tetrapeptide substrates, DEVD-pNA and IETD-pNA, respectively. The assay mixture consisted of 50 ll of midgut cells homogenate and 50 ll of chilled cell lysis buffer incubated on ice for 10 min. After incubation, 50 ll of 2 reaction buffer (containing 10 mM DTT) with 200 lM substrate (DEVD-pNA for Drice, and IETD-pNA for Dronc) was added and incubated at 37 °C for 1.5 h. The reaction was quantified at 405 nm.
168
2.8. Assay to detect apoptosis
181
The apoptotic cells were analyzed by staining with ethidium bromide (EB) and acridine orange (AO). The midguts of the larvae were collected in PSS. The PSS was replaced by 300 ll of collagenase (0.5 mg/ml) and kept for 15 min at 25 °C. The collagenase was removed and the pellet was washed three times by PBS with gentle shaking [27]. Finally the pellet was suspended in 80 ll of PBS. About 25 ll of cell suspension was mixed with 2 ll of EB/AO dye. The staining dye was prepared by dissolving 100 lg/ml AO and 100 lg/ml EB in PBS. About 100 cells were scored per treatment (5 replicates/group) for estimating the apoptotic index and expressed in percent [28].
182
Please cite this article in press as: R. Sachdev et al., Evaluation of the toxic potential of cefotaxime in the third instar larvae of transgenic Drosophila melanogaster, Chemico-Biological Interactions (2015), http://dx.doi.org/10.1016/j.cbi.2015.03.004
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2.9. Analysis of DNA damage by comet assay
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209
The comet assay was performed according to Mukhopadhyay et al. [27]. The midguts from 20 larvae were explanted in PSS. PSS in microcentrifuge tube was replaced by 300 ll of collagenase (0.5 mg/ml in PBS, pH 7.4) and kept for 15 min at 25 °C. The cell suspension was prepared by washing three times in PBS and finally the cells were suspended in 80 ll of PBS. The cell viability was checked by performing trypan blue assay before beginning the experiment [29]. Slides were stained with ethidium bromide (20 lg/ml; 75 ll/slides) for 10 min in dark. The slides were then dipped in chilled distilled water to remove the excess of stain and subsequently cover slips were placed over them. Each experiment was performed in triplicate and the slides were prepared in duplicate. Twenty-five cells per slide were randomly captured at a constant depth of the gel, and mean tail length was calculated to measure DNA damage by using comet score 1.5 software (TriTek Corporation).
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2.10. Statistical analysis
211 213
All data were expressed as mean ± standard error and Dunnett ANOVA test was used for the analysis by using software SPSS 16. Statistical significance was considered at 5% level.
214
3. Results
215
The results of the present study reveal that the cefotaxime doses of 10 and 20 lg/ml did not show any toxic effects for 6, 12, 24 and 48 h of the exposure. The larvae exposed to 40, 60 and 80 lg/ml of cefotaxime for 12, 24 and 48 h of duration showed a dose and duration dependent significant increase in the activity of b-galactosidase as compared to untreated (Fig. 1). The results obtained for in situ histochemical b-galactosidase activity for 48 h of exposure to 40, 60 and 80 lg/ml of cefotaxime are shown in Fig. 2(A–D). A dose dependent blue staining was observed in the proventriculus, foregut, midgut and brain ganglia (Fig. 2B–D). No blue staining was observed in untreated larvae (Fig. 2A). For the trypan blue exclusion assay, the larvae exposed to 10 and 20 lg/ml did not show any tissue damage for the 6, 12, 24 (Fig. 3B and C) and 48 h (Fig. 3D and E) of the exposure The larvae exposed to 40, 60 and 80 lg/ml of cefotaxime for 24 h showed a slight dose dependent tissue damage in the salivary glands, foregut and midgut regions. The larvae exposed to 40, 60 and 80 lg/ml of cefotaxime for 48 h of duration showed a dose
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3
dependent tissue damage in the salivary glands, foregut and midguts (Fig. 3F and G). The results obtained for lipid peroxidation assay are shown in Fig. 4. A significant dose and duration dependent increase in the mean absorbance value was observed in the larvae exposed to 40, 60 and 80 lg/ml after 12, 24 and 48 h compared to untreated (Fig. 4).The results obtained for total protein content are expressed in Fig. 5. The larvae exposed to 40, 60 and 80 lg/ml of cefotaxime for 24 and 48 h of duration showed a dose and duration dependent decrease in the total protein content (Fig. 5). The results obtained for GSH content are shown in Fig. 6. A dose and duration dependent significant decrease compared to untreated in the GSH content was observed for 12, 24 and 48 h of exposure of larvae to 40, 60 and 80 lg/ml of cefotaxime (Fig. 6). A dose and duration dependent significant increase compared to untreated in the GST level was observed in the larvae exposed to 40, 60 and 80 lg/ml of cefotaxime for 12, 24 and 48 h (Fig. 7). The results obtained for protein carbonyl content are shown in Fig. 8. A significant dose and duration dependent increase was observed in PC content among the group of larvae exposed to 40, 60 and 80 lg/ml of cefotaxime for 24 as well as 48 h (Fig. 8). Figs. 9 and 10 shows the relative intensities of the cleaved chromophore (p-nitroanilide) obtained after specific cleavage by caspases. A significant increase in the activity of caspase-3 was observed in the larvae exposed to 40, 60 and 80 lg/ml of cefotaxime for 24 and 48 h (Fig. 9). A similar trend was observed for caspase-9 activity in the larvae exposed to 40, 60 and 80 lg/ml of cefotaxime for 24 and 48 h (Fig. 10). For the apoptotic index analysis the normal and apoptotic midgut cells of the larvae are shown in Fig. 11(A and B). A significant dose and duration dependent increase in the apoptotic index was observed in the larvae exposed to 40, 60 and 80 lg/ml of cefotaxime after 24 and 48 h (Fig. 11C). The comet assay performed in the midgut cells of the larvae exposed to cefotaxime is shown in Fig. 12(A and B). No significant increase in the mean tail length was observed in the midgut cells of the larvae exposed to 10 and 20 lg/ml of cefotaxime for 6, 12, 24 and 48 h of duration (Fig. 12C). The larvae exposed to 40, 60 and 80 lg/ml of cefotaxime for 24 and 48 h of duration showed a dose and duration dependent significant increase in the mean tail length (Fig. 12C).
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4. Discussion
274
Various toxicological studies performed on animals showed that the cefotaxime is well tolerated by animals. Cefotaxime did not show any mutagenic or teratological effects in animals [4].
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Fig. 1. b-Galactosidase activity measured in transgenic Drosophila melanogaster (hsp70-lacZ)Bg9 third instar larvae exposed to different doses of cefotaxime for various durations (⁄p
No. of Pages 10, Model 5G
11 March 2015 Chemico-Biological Interactions xxx (2015) xxx–xxx 1
Contents lists available at ScienceDirect
Chemico-Biological Interactions journal homepage: www.elsevier.com/locate/chembioint 5 6
4
Evaluation of the toxic potential of cefotaxime in the third instar larvae of transgenic Drosophila melanogaster
7
Rahul Sachdev, Smita Jyoti, Falaq Naz, Yasir Hasan Siddique ⇑
8
Drosophila Transgenic Laboratory, Section of Genetics, Department of Zoology, Faculty of Life Sciences, Aligarh Muslim University, Aligarh, Uttar Pradesh 202002, India
3
10 9 11 1 2 3 6 14 15 16 17 18 19 20 21 22 23 24 25
a r t i c l e
i n f o
Article history: Received 23 July 2014 Received in revised form 25 December 2014 Accepted 3 March 2015 Available online xxxx Keywords: Tissue damage Oxidative stress Comet assay b-Galactosidase Drosophila melanogaster (hsp70-lacZ)Bg9
a b s t r a c t The present study was carried out to evaluate the toxic potential of cefotaxime in the third instar larvae of transgenic Drosophila melanogaster (hsp70-lacZ)Bg9. Cefotaxime at final concentration of 10, 20, 40, 60 and 80 lg/ml was mixed in the diet and the larvae were exposed to the selected doses for 6, 12, 24, 48 h. The hsp70 expression, trypan blue exclusion test, in situ histochemical b-galactosidase activity, lipid peroxidation, total protein content, glutathione (GSH) content, glutathione-S-transferase (GST) activity, protein carbonyl content, caspase 3 and 9 activity, apoptotic index and comet assay were taken as parameters for the study. The larvae exposed to 40, 60 and 80 lg/ml for 12, 24 and 48 h showed a dose and duration dependent significant increase in the activity of b-galactosidase and lipid peroxidation but decrease in the total GSH content as compared to unexposed larvae. The decrease in protein content was observed in the larvae exposed to 40, 60 and 80 lg/ml of cefotaxime for 24 and 48 h. The larvae exposed to 40, 60 and 80 lg/ml of cefotaxime for 24 and 48 h showed a dose and duration dependent increase in the tissue damage, GST, caspase 3 and 9 activity, PC content, apoptosis and the DNA tail length (comet assay). The result suggests that the cefotaxime is toxic at 40, 60 and 80 lg/ml of doses for the third instar larvae of transgenic D. melanogaster (hsp70-lacZ)Bg9. Cefotaxime at 10 and 20 lg/ml was not toxic for any duration of exposure. Ó 2015 Published by Elsevier Ireland Ltd.
27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44
45 46
1. Introduction
47
Cefotaxime (CTX), a third generation broad spectrum cephalosporin is commonly used as perioperative anti-microbial prophylactic in orthotropic liver transplantation (OLT) [1]. It provides protection against wide range of bacteria [2]. It has been reported for the adverse effects in humans showing an increase in serum aspartate amino transferase level and the occurrence of pseudomembranous colitis [3]. It has been reported for extremely low toxicity profile with an intravenous LD50 values in the range of 9–10 g/kg [4]. The higher doses of cefotaxime (600 mg/kg/day) showed a significant mortality in neonatal and adult mice [5]. Antibiotics have been used in therapeutics for more than 70 years, though no rational standardized approaches have been defined for the treatment in humans [6]. Due to ethical reasons, the toxic evaluations cannot be performed directly on humans. The fruit fly Drosophila melanogaster has been extensively used in basic and applied research for various toxicological evaluations and human disorders [7–11]. Fly has advantage over mammalian model because of the ease of laboratory maintenance and genetic
48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64
⇑ Corresponding author. E-mail address: [email protected] (Y.H. Siddique).
manipulations. As an alternative to higher animal, Drosophila has been well documented and recommended for toxicological evaluations. About 50% of the fly protein sequences have mammalian homologs and the results obtained can be extrapolated by using various mathematical models [12]. Heat shock proteins (HSPs) were initially reported to be expressed in response to heat but their expression is also triggered in response to diverse range of stress [13]. In the recent years, hsp70 has been considered to be one of the candidate genes for predicting cytotoxicity against environmental chemicals [14]. The present study was conducted to evaluate the toxic potential of cefotaxime using hsp70 expression, tissue damage, X-gal staining, lipid peroxidation, protein content, apoptosis and comet assay as parameters in the third instar larvae of transgenic D. melanogaster (hsp70-lacZ)Bg9 at various doses and durations of exposure.
65
2. Materials and methods
80
2.1. Fly strain
81
A transgenic D. melanogaster line expressing bacterial b-galactosidase in response to stress was used in the present study [15].
82
http://dx.doi.org/10.1016/j.cbi.2015.03.004 0009-2797/Ó 2015 Published by Elsevier Ireland Ltd.
Please cite this article in press as: R. Sachdev et al., Evaluation of the toxic potential of cefotaxime in the third instar larvae of transgenic Drosophila melanogaster, Chemico-Biological Interactions (2015), http://dx.doi.org/10.1016/j.cbi.2015.03.004
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In the afore mentioned strain, the transformation vector is inserted with a P-element, i.e., the line contains wild type hsp70 sequence up to lacZ fusion point. The flies and larvae were cultured on standard Drosophila food containing agar, corn meal, sugar and yeast at 24 ± 1 °C [16,17].
89
2.2. Experimental design
84 85 86 87
92
The final concentrations of cefotaxime, i.e., 10, 20, 40, 60 and 80 lg/ml were established in diet. The third instar larvae were allowed to feed for 6, 12, 24 and 48 h.
93
2.3. Soluble O-nitrophenyl-b-D-galactopyranoside (ONPG assay)
94
The expression of hsp70 was quantified by performing soluble O-nitrophenyl-b-D-galactopyranoside (ONPG) assay as described by Nazir et al. [16]. After giving a wash of phosphate buffer, the larvae were placed in microcentrifuge tubes (20 larvae/tube; 5 replicates/group), permeabilized for 10 min by acetone and incubated overnight at 37 °C in 600 ll of ONPG buffer. After incubation for the desired duration the reaction was stopped by adding 300 ll of Na2CO3 and the extent of the reaction was quantified by measuring absorbance at 420 nm [18,19].
90 91
95 96 97 98 99 100 101 102
103
2.4. In situ histochemical b-galactosidase activity
104
The larvae (10 larvae/treatment; 5 replicates/group) were dissected out in Pole’s salt solution (PSS) and X-gal staining was performed using the method as described by Chowdhuri et al. [18]. The larvae explants were fixed in 2.5% glutaraldehyde, washed in 50 mM sodium phosphate buffer (pH 8.0) and stained overnight in X-gal staining solution at 37 °C in the dark.
105 106 107 108 109
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2.5. Trypan blue exclusion test
111
For studying the extent of tissue damage in larvae caused by the exposure to different dosages of cefotaxime dye exclusion test was performed [16,20]. Briefly, the internal tissues of larvae were explanted in a drop of PSS, washed in phosphate buffer saline (PBS), stained in trypan blue (0.2 mg/ml in PBS) for 30 min, washed thoroughly in PBS, and scored immediately for dark blue staining. About 50 larvae per treatment (10 larvae per dose; 5 replicates group) were scored for the trypan blue staining on an average composite index per larvae: no color = 0; any blue = 1; darkly stained = 2; large patches of darkly stained cells = 3; or complete staining of most cells in the tissue = 4 [20].
112 113 114 115 116 117 118 119 120 121
122
2.6. Preparation of larval homogenate
123
The larvae (10 larvae/dose; 5 replicates/group) were homogenized in 1 ml of cold homogenizing buffer (0.1 M phosphate buffer containing 0.15 M KCl; pH 7.4). The supernatant after centrifugation at 9000g was used for estimating lipid peroxidation, total protein, glutathione content, glutathione-S-transferase activity and protein carbonyl content.
124 125 126 127 128
129 130 131
132 133 134 135
2.6.1. Lipid peroxidation assay Lipid peroxidation assay was performed as described earlier using 1,1,3,3-tetramethoxy propane as a standard [21,22]. 2.6.2. Protein estimation The protein content in all the treated as well as untreated groups were estimated according to the method of Bradford (1976) using bovine serum albumin (BSA) as a standard [23].
2.6.3. Estimation of glutathione (GSH) content The glutathione (GSH) content was estimated colorimetrically using Ellman’s reagent (DTNB) according to the procedure described by [24]. The supernatant was precipitated with 4% sulphosalicylic acid (4%) in the ratio of 1:1. The samples were kept at 4 °C for 1 h and then subjected to centrifugation at 5000 rpm for 10 min at 4 °C. The assay mixture consisted of 550 ll of 0.1 M phosphate buffer, 100 ll of supernatant and 100 ll of DTNB. The OD was read at 412 nm and the results were expressed as l moles of GSH/gram tissue.
136
2.6.4. Estimation of glutathione-S-transferase (GST) activity The glutathione-S-transferase activity was determined by the method of Habig et al. [25]. The reaction mixture consist of 500 ll of 0.1 M phosphate buffer, 150 ll of 10 mM CDNB, 200 ll of 10 mM reduced glutathione and 50 ll of supernatant. The OD were take at 340 nm and the enzyme activity was expressed as l moles of CDNB conjugates/min/mg protein.
146
2.6.5. Estimation of protein carbonyl content The protein carbonyl content was estimated according to the protocol described by Hawkins et al. [26]. The larvae homogenate was diluted to a protein concentration of approx. 1 mg/ml. About 250 ll of each diluted homogenate was taken in Eppendorf centrifuge tubes separately. To it 250 ll of 10 mM 2,4-dinitrophenyl hydrazine (dissolved in 2.5 M HCl) was added, vortexed and kept in dark for 20 min. About 125 ll of 50% (w/v) trichloroacetic acid (TCA) was added, mixed thoroughly and incubated at 20 °C for 15 min. The tubes were then centrifuged at 4 °C for 10 min at 9000 rpm. The supernatant was discarded and the pellet obtained was washed twice by ice cold ethanol:ethyl acetate (1:1). Finally the pellets were re-dissolved in 1 ml of 6 M guanidine hydrochloride and the absorbance was read at 370 nm.
153
2.7. Assay for caspase-3 (Drice) and caspase-9 (Dronc) activities
167
For estimating the activities of caspase 3 and 9 the midguts of the larvae were collected in PBS. The assay was performed according to the manufacturer protocol with some modification (Bio-Vision, CA, USA). The assay was based on spectrophotometric detection of the chromophore p-nitroanilide (pNA) obtained after specific action of caspase-3 and caspase-9 on tetrapeptide substrates, DEVD-pNA and IETD-pNA, respectively. The assay mixture consisted of 50 ll of midgut cells homogenate and 50 ll of chilled cell lysis buffer incubated on ice for 10 min. After incubation, 50 ll of 2 reaction buffer (containing 10 mM DTT) with 200 lM substrate (DEVD-pNA for Drice, and IETD-pNA for Dronc) was added and incubated at 37 °C for 1.5 h. The reaction was quantified at 405 nm.
168
2.8. Assay to detect apoptosis
181
The apoptotic cells were analyzed by staining with ethidium bromide (EB) and acridine orange (AO). The midguts of the larvae were collected in PSS. The PSS was replaced by 300 ll of collagenase (0.5 mg/ml) and kept for 15 min at 25 °C. The collagenase was removed and the pellet was washed three times by PBS with gentle shaking [27]. Finally the pellet was suspended in 80 ll of PBS. About 25 ll of cell suspension was mixed with 2 ll of EB/AO dye. The staining dye was prepared by dissolving 100 lg/ml AO and 100 lg/ml EB in PBS. About 100 cells were scored per treatment (5 replicates/group) for estimating the apoptotic index and expressed in percent [28].
182
Please cite this article in press as: R. Sachdev et al., Evaluation of the toxic potential of cefotaxime in the third instar larvae of transgenic Drosophila melanogaster, Chemico-Biological Interactions (2015), http://dx.doi.org/10.1016/j.cbi.2015.03.004
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2.9. Analysis of DNA damage by comet assay
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The comet assay was performed according to Mukhopadhyay et al. [27]. The midguts from 20 larvae were explanted in PSS. PSS in microcentrifuge tube was replaced by 300 ll of collagenase (0.5 mg/ml in PBS, pH 7.4) and kept for 15 min at 25 °C. The cell suspension was prepared by washing three times in PBS and finally the cells were suspended in 80 ll of PBS. The cell viability was checked by performing trypan blue assay before beginning the experiment [29]. Slides were stained with ethidium bromide (20 lg/ml; 75 ll/slides) for 10 min in dark. The slides were then dipped in chilled distilled water to remove the excess of stain and subsequently cover slips were placed over them. Each experiment was performed in triplicate and the slides were prepared in duplicate. Twenty-five cells per slide were randomly captured at a constant depth of the gel, and mean tail length was calculated to measure DNA damage by using comet score 1.5 software (TriTek Corporation).
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2.10. Statistical analysis
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All data were expressed as mean ± standard error and Dunnett ANOVA test was used for the analysis by using software SPSS 16. Statistical significance was considered at 5% level.
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3. Results
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The results of the present study reveal that the cefotaxime doses of 10 and 20 lg/ml did not show any toxic effects for 6, 12, 24 and 48 h of the exposure. The larvae exposed to 40, 60 and 80 lg/ml of cefotaxime for 12, 24 and 48 h of duration showed a dose and duration dependent significant increase in the activity of b-galactosidase as compared to untreated (Fig. 1). The results obtained for in situ histochemical b-galactosidase activity for 48 h of exposure to 40, 60 and 80 lg/ml of cefotaxime are shown in Fig. 2(A–D). A dose dependent blue staining was observed in the proventriculus, foregut, midgut and brain ganglia (Fig. 2B–D). No blue staining was observed in untreated larvae (Fig. 2A). For the trypan blue exclusion assay, the larvae exposed to 10 and 20 lg/ml did not show any tissue damage for the 6, 12, 24 (Fig. 3B and C) and 48 h (Fig. 3D and E) of the exposure The larvae exposed to 40, 60 and 80 lg/ml of cefotaxime for 24 h showed a slight dose dependent tissue damage in the salivary glands, foregut and midgut regions. The larvae exposed to 40, 60 and 80 lg/ml of cefotaxime for 48 h of duration showed a dose
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dependent tissue damage in the salivary glands, foregut and midguts (Fig. 3F and G). The results obtained for lipid peroxidation assay are shown in Fig. 4. A significant dose and duration dependent increase in the mean absorbance value was observed in the larvae exposed to 40, 60 and 80 lg/ml after 12, 24 and 48 h compared to untreated (Fig. 4).The results obtained for total protein content are expressed in Fig. 5. The larvae exposed to 40, 60 and 80 lg/ml of cefotaxime for 24 and 48 h of duration showed a dose and duration dependent decrease in the total protein content (Fig. 5). The results obtained for GSH content are shown in Fig. 6. A dose and duration dependent significant decrease compared to untreated in the GSH content was observed for 12, 24 and 48 h of exposure of larvae to 40, 60 and 80 lg/ml of cefotaxime (Fig. 6). A dose and duration dependent significant increase compared to untreated in the GST level was observed in the larvae exposed to 40, 60 and 80 lg/ml of cefotaxime for 12, 24 and 48 h (Fig. 7). The results obtained for protein carbonyl content are shown in Fig. 8. A significant dose and duration dependent increase was observed in PC content among the group of larvae exposed to 40, 60 and 80 lg/ml of cefotaxime for 24 as well as 48 h (Fig. 8). Figs. 9 and 10 shows the relative intensities of the cleaved chromophore (p-nitroanilide) obtained after specific cleavage by caspases. A significant increase in the activity of caspase-3 was observed in the larvae exposed to 40, 60 and 80 lg/ml of cefotaxime for 24 and 48 h (Fig. 9). A similar trend was observed for caspase-9 activity in the larvae exposed to 40, 60 and 80 lg/ml of cefotaxime for 24 and 48 h (Fig. 10). For the apoptotic index analysis the normal and apoptotic midgut cells of the larvae are shown in Fig. 11(A and B). A significant dose and duration dependent increase in the apoptotic index was observed in the larvae exposed to 40, 60 and 80 lg/ml of cefotaxime after 24 and 48 h (Fig. 11C). The comet assay performed in the midgut cells of the larvae exposed to cefotaxime is shown in Fig. 12(A and B). No significant increase in the mean tail length was observed in the midgut cells of the larvae exposed to 10 and 20 lg/ml of cefotaxime for 6, 12, 24 and 48 h of duration (Fig. 12C). The larvae exposed to 40, 60 and 80 lg/ml of cefotaxime for 24 and 48 h of duration showed a dose and duration dependent significant increase in the mean tail length (Fig. 12C).
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4. Discussion
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Various toxicological studies performed on animals showed that the cefotaxime is well tolerated by animals. Cefotaxime did not show any mutagenic or teratological effects in animals [4].
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Fig. 1. b-Galactosidase activity measured in transgenic Drosophila melanogaster (hsp70-lacZ)Bg9 third instar larvae exposed to different doses of cefotaxime for various durations (⁄p
