Exposure to streptomycin alters oxidative and antioxidative response in larval midgut tissues of Galleria mellonella

Exposure to streptomycin alters oxidative and antioxidative response

in larval midgut tissues of Galleria mellonella

Ender Büyükgüzel

, Yusuf Kalender

a

Department of Biology, Faculty of Arts and Science, Karaelmas University, 67100 Zonguldak, Turkey

b_{Department of Biology, Faculty of Arts and Science, Gazi University, 06100 Ankara, Turkey}

a r t i c l e

i n f o

Article history:

Received 4 February 2008 Accepted 30 April 2009 Available online 6 May 2009

Keywords: Galleria mellonella Streptomycin Antioxidant enzymes Transaminases Midgut Malondialdehyde

a b s t r a c t

Although antibiotics have different molecular modes of actions, increasing evidence for their secondary effects suggests that they disturb cellular homeostasis by generating free radical intermediates that trig-ger lipid peroxidation, which leads to oxidative stress. Streptomycin is an antibiotic insecticide used to control pest insects and microbial diseases of agricultural crops. We investigated the biochemical basis for pro-oxidative effects of streptomycin in the midgut tissues of greater wax moth, Galleria mellonella (L.) seventh-instar larvae by measuring content of the oxidative stress indicator, malondialdehyde (MDA), and antioxidant enzymes [superoxide dismutase (SOD), catalase (CAT), glutathione S-transferase (GST) and glutathione peroxidase (GPx)] and transaminases [alanine aminotransferase (ALT), aspartate aminotransferase (AST)] activities. The insects were reared from first-instar larvae on artificial diets con-taining 0.001, 0.01, 0.1 or 1.0 g streptomycin per 100 g of diets. The supplementation of streptomycin at high concentrations to the diets caused oxidative stress as evidenced by the elevation of MDA content, SOD and GPx activities, accompanied by the concurrent depletion of CAT and GST activities. The strepto-mycin-induced oxidative stress was also accompanied by decreases of transaminases activities in midgut tissues. We found a significant negative correlation of MDA contents with GST activities in the larval mid-gut tissues. These results suggest that exposure to dietary streptomycin resulted in oxidative stress which could impact midgut digestive physiology at the expense of impairment of antioxidant and transami-nases enzymes in G. mellonella larvae.

Ó 2009 Elsevier Inc. All rights reserved.

1. Introduction

Greater wax moth, Galleria mellonella (L.) is a pyralid moth whose larvae feed on combs, wax and honey in beehives. Because of this feeding regime, this insect is major pest that damages hive products and causes economical losses. Some fumigants, such as phosphine gas and methyl bromide which are highly toxic to non-targets, have been commonly used to control greater wax moth and lesser wax moth in stored hive products[1]and other pest in-sects of stored products[2]. Concerns over the adverse effects of the continued use of conventional pesticides and fumigants on hu-man health and the environment, together with continuing prob-lems of pest resistance, have encouraged the search for safer and environmentally sustainable alternatives for pest management.

Although some conventional antibiotic insecticides such as aba-mectin have been used for chemical pest management, their use is restricted because of their toxicity to nontargets[3]. Attention has been given to clinically important antibiotics in controlling pest

in-sects as their nonmedical use. Interest in these antibiotics includ-ing streptomycin comes from studies to determine appropriate dietary levels to preserve insect culture media from microbial con-tamination and ensure development of insect up to adult emer-gence. Although culturing insects in the presence of dietary antibiotics is a decades-old practice, antibiotics at concentrations above ‘‘safe” levels can exert deleterious influences on insects[4– 12].

An aminoglycoside, streptomycin is a clinically important anti-biotic used to treat bacterial infections of human and animals[13]. It is also of importance in agricultural purposes as pesticide to con-trol pest insects and microbial disease of agricultural crops

[5,7,11,12,14,15]. We have recorded that streptomycin at high con-centrations decreased survivorship, altered body mass and chemi-cal composition, and retarded development of G. mellonella[10]. Many antibiotics exert their bactericidal effects via the production of hydroxyl radical (HO

) [16]and other reactive oxygen species (ROS)[17,18]regardless of their molecular targets. Hydroxyl radi-cal is particularly capable of inducing oxidative damage to cells by lipid peroxidation as well as damage to proteins and DNA in hu-man and animal tissues[19]. A recent study by Kohenski et al.

[20] presented an evidence that despite the diversity of their 0048-3575/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved.

doi:10.1016/j.pestbp.2009.04.008

*Corresponding author. Karaelmas University, Faculty of Arts and Science,

Department of Biology, 67100 Incıvez-Zonguldak, Turkey. Fax: +90 372 2574181.

E-mail address:endericen@hotmail.com(E. Büyükgüzel).

Contents lists available atScienceDirect

Pesticide Biochemistry and Physiology

action, three bactericidal antibiotics, ampicillin, kanamycin and norfloxacin stimulated overproduction of the ROS, HO

and intra-cellular accumulation of fluorescent hydroxyphenyl fluorescein which ultimately contribute to cell death. However, there is also a conflicting report that none of the commonly used antibiotics at pharmalogical concentrations affect the oxidative status in pancre-atic islet cell viability except for a slight increase in malondialde-hyde (MDA) and nitric oxide (NO) levels with some antibiotics[21]. The midgut tissue of G. mellonella seventh-instars responds to penicillin-induced oxidative stress at the expense of up-regulation of antioxidative enzymes[22]. Lepidopteran larvae maintain opti-mal gut conditions while minimizing any deleterious effects of pro-oxidant metabolites. Much of the oxidative stress in midgut tissue results from oxidative injury attributed to hydrogen perox-ide due to dietary allelochemicals[23]. A portion of ROS is scav-enged by non-enzymatic antioxidants such as ascorbate, glutathione, tocopherols, and carotenoids[24]but most are elimi-nated by a suite of antioxidant enzymes in insects. ROS can cause oxidative stress and lead to uncontrolled lipid peroxidation, pro-tein, enzyme, DNA oxidation in insect tissues[25,26]. Overproduc-tion of ROS impairs the absorpOverproduc-tion of ingested nutrient and can cause oxidative damage to the midgut cells rendering the digestive tract nonfunctional[26]. The oxidative destruction of lipids acts in a chain reaction to form lipid hydroperoxides which can decom-pose to MDA and 4-hydroxynonenal (4HNE) as end products

[27]. MDA, the quantitatively predominant aldehyde, forms Schiff bases with amines of proteins, phospholipids, and nucleic acids leading to damaged cellular biomolecules. Insect antioxidant en-zymes include superoxide dismutase (SOD), catalase (CAT), gluta-thione peroxidase (GPx) and glutagluta-thione S-transferases (GST)

[24,28]. GST has a wide range of substrate specificities among anti-oxidant enzymes involved in detoxification of xenobiotics [29]. GPx (selenium-independent) metabolize H2O2and deleterious

li-pid peroxides using reduced glutathione as a substrate[30]. SOD catalyzes the dismutation of superoxide radicals to H2O2and

oxy-gen and appears to be the main response to dietary pro-oxidant exposure[31]. CAT reduces H2O2to water and oxygen[32]. It has

also been recorded that antibiotics impaired alanine aminotrans-ferase (ALT) and aspartate aminotransaminotrans-ferase (AST) enzymes activi-ties indicating cellular disfunctions due to increased oxidative stress in midgut tissue of seventh-instar larvae of G. mellonella

[22]as well as in silk gland of Philosamia ricini (Boisduval) last in-star larvae[33]. Transaminases supply substrates for the tricarbox-ylic acid cycle via amino acid metabolism to interconvert energy storage molecules in midgut and other lepidopteran tissues[34]. We surmise transaminases along with antioxidant enzymes are key biomarkers in assessing oxidative tissue injury due to antibi-otic toxicity in insects. We investigated the hypothesis that supple-menting of streptomycin to G. mellonella diet produces oxidative stress in the larval midgut tissue. This stress leads to crippled anti-oxidative defense systems and to metabolic dysfunction. In this pa-per we report on the outcomes of expa-periments designed to test our hypothesis.

2. Materials and methods 2.1. Insects

Larvae of the greater wax moth, G. mellonella were used in all experiments. The insects were reared in 1000-ml glass jars with an artificial diet[35]at 30 ± 1 °C in constant darkness. The stan-dard diet was composed of 420 g of bran, 150 ml of filtered honey, 150 ml of glycerol, 20 g of ground old dark honeycomb and 30 ml of distilled water. Fifteen newly emerged adult females were placed in the jars and provided a piece of old honeycomb on the

diet for egg deposition and feeding of newly hatched larvae. The methods used to prepare and dispense diets into containers, and the methods used to obtain eggs and larvae and their placement onto diets were described previously[10,22,36].

2.2. Experimental designs

Streptomycin sulfate (Sigma Chemical Co., St. Louis, USA) was directly incorporated into diets at concentrations of 0.001, 0.01, 0.1 or 1.0 g per 100 g of diets. First-instar larvae were constantly reared on streptomycin-containing diets from hatching to sev-enth-instars. Because streptomycin decomposes under light, the artificial diets containing streptomycin concentrations were kept in constant darkness except during a short daily observation period for maintaining constant concentrations as described elsewhere

[7,14]. Streptomycin was tested because it is the oldest and most common antimicrobial used in the larval diets for artificial rearing of various insects[4,11]. Tested concentrations of streptomycin are actual concentrations which are practically relevant in a range of 0.01–1.0% incorporated into commercial diets used for artificial rearing of insects[9,11,12]. Our preliminary experiments showed that these dietary concentrations enable larvae to complete their adult development with gradually increasing mortality. In the light of these observations, we used these actual concentrations for obtaining specific sublethal toxicity at high concentrations of streptomycin. Using standard laboratory rearing conditions, exper-iments were carried out to examine the effects of streptomycin on lipid peroxidation levels, and activities of antioxidant and transam-inases enzymes, in midgut of seventh-instars. Newly molted larvae in this instar was recognized by the size of the head capsules[37]. Seventh-instars, which are active feeding stage, was used to deter-mine effects of streptomycin on oxidative and antioxidative re-sponse in larval midgut tissues because our preliminary feeding experiments demonstrated that streptomycin exerted its adverse effects on mostly larval stages of the insect.

2.3. Tissue collection

Seventh-instars were used to determine the lipid peroxidation product, MDA content, and antioxidant enzyme activities. The lar-vae were chilled on ice for 5 min and surface sterilized in 95% eth-anol. The larvae were then cut longitudinally before the first pair of thoracic legs and behind the third pair of abdominal appendages using dissection scissors and the midguts were withdrawn with fine-tipped forceps under stereo-microscope (Olympus SZ61, To-kyo, Japan). Adhering fat body, malpighian tubules and gut con-tents were then removed. Midguts were collected into a chilled Eppendorf tube charged with cold homogenization buffer [w/v 1.15% KCl, 25 mM K2HPO4, 5 mM ethylen-diaminetetraacetic acid

(EDTA), 2 mM phenylmethylsulphonyl fluoride (PMSF), 2 mM dithiotreitol (DTT), pH 7.4] and stored at 80 °C. The cryotubes were kept at room temperature until the tissue began to thaw be-fore using.

2.4. Sample preparation

Extracts of midgut were prepared at 4 °C by an ultrasonic homogenizer (Bandelin Sonoplus, HD2070, Berlin, Germany) at 30 W, 10 s in homogenization buffer and subsequent centrifuga-tion (Hettich Zentrifugen Mikro200R, Germany) at 10,000g for 15 min at 4 °C. The resulting cell-free extracts were collected for biochemical analysis. Supernatants were centrifuged at either 1000g for 10 min at 4 °C (SOD and CAT assays), 16,000g for 20 min (GST, GPx, ALT and AST) or 2000g for 15 min (lipid peroxi-dation). MDA contents and antioxidant enzymes activities were determined by measuring the absorbance of the samples in a dual

beam spectrophotometer (Shimadzu 1700, UV/vis, Kyoto, Japan). Assays were replicated four times each with ten midguts. Protein concentrations were determined according to Lowry et al.[38]by using bovine serum albumin (BSA) as a standard. All chemicals used were analytical grade and were obtained from Sigma-Aldrich (St. Louis, MO, USA).

2.5. Measurement of malondialdehyde (MDA)

MDA is the most abundant individual aldehyde resulting from lipid peroxidation breakdown in biological systems and is com-monly used as an indirect index of lipid peroxidation. MDA con-tents were assayed with thiobarbituric (TBA) test according to Jain and Levine[39]. TBA test is an easy and quick assay for the assessment of lipid peroxidation in which MDA is derivatized. MDA reacts with TBA to form a colored complex. MDA contents as an indicator of lipid peroxidation were determined after incuba-tion at 95 °C with TBA (1% w/v). Absorbances were measured at 532 nm to determine MDA content. The amount of MDA formed was calculated, using a molar extinction coefficient of 1.56  105_M 1_cm 1_{and expressed as nmol/mg protein.}

2.6. Measurement of superoxide dismutase (SOD)

Total SOD (EC 1.15.1.1) activity was determined according to Marklund and Marklund[40]assaying the autooxidation and illumi-nation of pyrogallol at 440 nm for 3 min. One unit total SOD activity was calculated as the amount of protein causing 50% inhibition of pyrogallol autooxidation. The total SOD activity was expressed as units per milligram of protein (U mg 1_{). A blank without}

homoge-nate was used as a control for non-enzymatic oxidation of pyrogallol in Tris-EDTA buffer (50 mM Tris, 10 mM EDTA, pH 8.2).

2.7. Measurement of catalase (CAT)

Before the determination of CAT (EC 1.11.1.6) activity, samples were diluted with 1:9 with 1% v/v Triton X-100. Enzyme activity was measured according to Aebi[41] assaying the hydrolysis of H2O2 and decreasing absorbance at 240 nm over a 3 min period

at 25 °C. CAT activity was expressed as millimoles of H2O2reduced

per minute per milligram of protein, using an extinction coefficient of 0.0394 mM 1cm 1. A blank without homogenate was used as a control for non-enzymatic hydrolysis of peroxide in phosphate buffer (50 mM, pH 7.0).

2.8. Measurement of glutathione-S-transferase (GST)

GST (EC 2.5.1.18) activity was assayed by measuring the forma-tion of the GSH and 1-chloro-2,4-dinitrobenzene (CDNB) conjugate

[42]. The increase in absorbance was recorded at 340 nm for 3 min. The specific activity of GST was expressed as

l

con-jugation using a corresponding substrate 25 mM CDNB and 20 mM GSH in in 50 mM phosphate buffer, pH 7.0.

2.9. Measurement of glutathione peroxidase (GPx)

GPx (EC 1.11.1.9) activity was measured with H2O2as substrate

according to Paglia and Valentine[43]. This reaction was moni-tored indirectly as the oxidation rate of NADPH at 340 nm for 3 min. Enzyme activity was expressed as nmol of NADPH con-sumed per minute per milligram of protein, using an extinction coefficient of 6.220 M 1_cm 1_{. A blank without homogenate was}

used as a control for the non-enzymatic oxidation of NADPH upon addition of hydrogen peroxide in Tris buffer (0.1 M, pH 8.0).

2.10. Measurement of alanine and aspartate aminotransferase (ALT and AST)

Aminotransferases activities were determined using Biomereux (Marcy I’Etoile, France) diagnostic kits [ALT (Kit No: 63313; EC 2.6.1.2, L-alanine 2-oxoglutarate aminotransferase) and AST (Kit

No: 63411; EC 2.6.1.1,L-aspartate 2-oxoglutarate

aminotransfer-ase)],L-aspartate and 2-oxoglutarate for AST,L-alanine and

2-oxo-glutarate for ALT were provided as substrates. ALT and AST activities were determined following manufacturer’s instructions provided in the kits. All assays were corrected for non-enzymatic reactions using corresponding substrate in phosphate buffer (50 mM, pH 7.0).

2.11. Statistical analysis

We used one-way analysis of variance (ANOVA) [44] with four replicated blocks to compare within treatments factors (diets with different concentrations of streptomycin vs controls) for MDA content, antioxidant enzymes and transaminases activ-ities. To determine significant differences between means, least significant differences (LSD) [44] test was used. When the F estimate exceeded the probability of 0.05 the differences were considered significant. Regression analysis was also performed to test the correlation between MDA content and antioxidant enzymes activities[44].

3. Results

3.1. Malondialdehyde content

The diet containing high streptomycin concentrations signifi-cantly increased midgut MDA content. The diet amended with low antibiotic concentrations (0.001 and 0.01 g) did not signifi-cantly affect MDA content (Fig. 1).

3.2. Antioxidant enzymes

SOD activity was significantly decreased by low streptomycin concentrations. However, the midgut enzyme activity at the high-est antibiotic concentration (1.0 g) was significantly increased (Fig. 2A). The 0.01, 0.1 and 1.0 g dietary antibiotic concentrations resulted in significantly decreased CAT activity in the midgut of the larvae in comparison to control. However, the lowest dietary

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0 0.001 0.01 0.1 1.0 Streptomycin concentrations (g/100g)

MDA content (nmol/mg protein)

a,b,c

a,b,c,d

Fig. 1. Effects of dietary streptomycin on the content of MDA in midguts of larvae. (a) Comparison of control and all concentrations of streptomycin (P < 0.05). (b) Comparison of 0.001 g of streptomycin with 0.01, 0.1 and 1.0 g of streptomycin (P < 0.05). (c) Comparison of 0.01 g of streptomycin with 0.1 and 1.0 g of streptomycin (P < 0.05). (d) Comparison of 0.1 g of streptomycin with 1.0 g of streptomycin (P < 0.05). Bars represent the means (±SE) of four replicates with 10 midguts per replicate.

antibiotic concentration (0.001 g) did not affect the enzyme activ-ity (Fig. 2B). Low dietary streptomycin concentrations (0.001 and 0.01 g) resulted in significantly increased midgut GST activities while the enzyme activities for 0.1 and 1.0 g streptomycin were

de-creased (Fig. 2C). The lowest dietary streptomycin concentration caused a significant decrease in GPx activity while 0.01 and 1.0 g antibiotic concentrations increased the enzyme activity in the mid-gut tissue in comparison to control (Fig. 2D).

0 0.002 0.004 0.006 0.008 0.01 0.012 0 0.001 0.01 0.1 1.0 Streptomycin concentrations (g/100g)

SOD activity (U/mg protein)

a a,b a,c a,b,c,d 0 5 10 15 20 25 30 35 40 45 Streptomycin concentrations (g/100g)

CAT activity (mmol/mg protein/min)

a,b a,b,c a,b,d 0 0.001 0.01 0.1 1.0 0 0.5 1 1.5 2 2.5 0 0.001 0.01 0.1 1.0 Streptomycin concentrations (g/100g)

GST activity (µmol/mg protein/min)

a a,b a ,b , c a,b,c 0 0.05 0.1 0.15 0.2 0.25 Streptomycin concentrations (g/100g)

GPx activity (nmol/mg protein/ min)

a a,b c a,b,d 0 0.001 0.01 0.1 1.0

A

B

D

C

Fig. 2. (A) Effects of dietary streptomycin on the activities of SOD in the midgut tissue of larvae. (a) Comparison of control and all concentrations of streptomycin (P < 0.05). (b) Comparison of 0.001 g of streptomycin with 0.01, 0.1 and 1.0 g of streptomycin (P < 0.05). (c) Comparison of 0.01 g of streptomycin with 0.1 and 1.0 g of streptomycin (P < 0.05). (d) Comparison of 0.1 g of streptomycin with 1.0 g of streptomycin (P < 0.05). Bars represent the means (±SE) of four replicates with 10 midguts per replicate. (B) Effects of dietary streptomycin on the activities of CAT in the midgut tissue of larvae. (a) Comparison of control and all concentrations of streptomycin (P < 0.05). (b) Comparison of 0.001 g of streptomycin with 0.01, 0.1 and 1.0 g of streptomycin (P < 0.05). (c) Comparison of 0.01 g of streptomycin with 0.1 and 1.0 g of streptomycin (P < 0.05). (d) Comparison of 0.1 g of streptomycin with 1.0 g of streptomycin (P < 0.05). Bars represent the means (±SE) of four replicates with 10 midguts per replicate. (C) Effects of dietary streptomycin on the activities of GST in the midgut tissue of larvae. (a) Comparison of control and all concentrations of streptomycin (P < 0.05). (b) Comparison of 0.001 g of streptomycin with 0.01, 0.1 and 1.0 g of streptomycin (P < 0.05). (c) Comparison of 0.01 g of streptomycin with 0.1 and 1.0 g of streptomycin (P < 0.05). (d) Comparison of 0.1 g of streptomycin with 1.0 g of streptomycin (P < 0.05). Bars represent the means (±SE) of four replicates with 10 midguts per replicate. (D) Effects of dietary streptomycin on the activities of GPx in the midgut tissue of larvae. (a) Comparison of control and all concentrations of streptomycin (P < 0.05). (b) Comparison of 0.001 g of streptomycin with 0.01, 0.1 and 1.0 g of streptomycin (P < 0.05). (c) Comparison of 0.01 g of streptomycin with 0.1 and 1.0 g of streptomycin (P < 0.05). (d) Comparison of 0.1 g of streptomycin with 1.0 g of streptomycin (P < 0.05). Bars represent the means (±SE) of four replicates with 10 midguts per replicate.

0 0.05 0.1 0.15 0.2 0.25 0.3 0 0.001 0.01 0.1 1.0 Streptomycin concentrations (g/100g)

ALT activity (U/mg protein)

a a, b a,b,c a,b,d 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0 0.001 0.01 0.1 1.0 Streptomycin concentrations (g/100g) A S T a c ti v it y ( U /m g pr ot e in ) a _a,b b,c a,b,c,d

A

_B

Fig. 3. (A) Effects of dietary streptomycin on the activities of ALT in the midgut tissue of larvae. (a) Comparison of control and all concentrations of streptomycin (P < 0.05). (b) Comparison of 0.001 g of streptomycin with 0.01, 0.1 and 1.0 g of streptomycin (P < 0.05). (c) Comparison of 0.01 g of streptomycin with 0.1 and 1.0 g of streptomycin (P < 0.05). (d) Comparison of 0.1 g of streptomycin with 1.0 g of streptomycin (P < 0.05). Bars represent the means (±SE) of four replicates with 10 midguts per replicate. (B) Effects of dietary streptomycin on the activities of AST in the midgut tissue of larvae. (a) Comparison of control and all concentrations of streptomycin (P < 0.05). (b) Comparison of 0.001 g of streptomycin with 0.01, 0.1 and 1.0 g of streptomycin (P < 0.05). (c) Comparison of 0.01 g of streptomycin with 0.1 and 1.0 g of streptomycin (P < 0.05). (d) Comparison of 0.1 g of streptomycin with 1.0 g of streptomycin (P < 0.05). Bars represent the means (±SE) of four replicates with 10 midguts per replicate.

3.3. Aminotransferases activities

All dietary streptomycin treatments resulted in significantly de-creased ALT activity in the midgut tissue. A 50% of decrease in the enzyme activity was recorded by the highest antibiotic concentra-tion (Fig. 3A). The 0.001, 0.01 and 1.0 g antibiotic concentrations decreased AST activity in comparison to control. The highest con-centration caused a 50% of significant decrease in AST activity in the midgut of the larvae (Fig. 3B).

MDA content was negatively correlated with GST (R2_{= 0.84,}

P < 0.05) activity in the midgut tissue of the larvae exposed to streptomycin. Consistent with the analysis, elevated content of MDA caused by increasing concentrations of streptomycin tended to decrease GST activities in the larvae. The larvae reared on the highest streptomycin concentration (1.0 g) showed increased con-tent of MDA (from 0.29 ± 0.03 to 0.69 ± 0.04 nmol/mg protein) while they showed decreased GST (from 1.36 ± 0.04 to 0.72 ± 0.05

l

4. Discussion

Our recent study showed that G. mellonella larvae are generally more susceptible to some antifungal and antibacterial antibiotics including an aminoglycoside antibiotic, streptomycin, because development times, survivorship and biochemical composition in different developmental stages were more strongly affected[10]. We attempted to relate these detrimental effects to pro-oxidative actions of antibiotics [22], however the mechanism underlying their detrimental effects on biological and biochemical parameters of insects has not yet been fully elucidated. The present work indi-cates that supplementation of diets with streptomycin caused lipid peroxidation, leading to oxidative stress in the larval midgut tissue of a pest insect model, G. mellonella.

Antibiotics have been reported to generate reactive oxygen rad-icals leading to oxidative stress which causes severe damage to cell membrane in various animal models [17–19,45]. In the present study, streptomycin-induced oxidative stress in the wax moth is confirmed by the elevation of midgut oxidized lipids, evidenced by significant increase in MDA and impaired antioxidant enzymes activities at high antibiotic doses suggesting a pro-oxidative effect of this antibiotic. Our suggestion is supported by findings of Mar-che et al.[46]who reported that some aminoglycoside antibiotics including streptomycin impair membrane phosphoinositide metabolism. These aminoglycoside antibiotics inhibit lysosomal phospholipases by binding to negatively charged phospholipid bilayers and this inhibition may be related to their pro-oxidant toxicity[47]. Some studies demonstrated that the early actions of some chemical and biological agents is mediated by calcium-mod-ulated activity of phospholipase A2(PLA2) in insects[48]and

high-er animal cells [49]. Moreover, streptomycin inhibits neurotransmission by interfering with calcium transport physiol-ogy at pre- or post-synaptic levels in insects[50]. It is therefore possible that streptomycin may interfere with cellular lipid metab-olism leading to generation of lipid hydroperoxides in the midgut of G. mellonella larvae. Free radicals have been shown to exhaust the antioxidant defense system and hence elevate the oxidation process of lipids in the foregut and midgut tissues of lepidopteran insect Spodoptera littoralis (Boisduval) [26]. The polyunsaturated fatty acids, which are precursors of eicosanoid and other related molecules[51], are essential for G. mellonella larvae to complete normal development and maintain physiological process [52]. The apparent dependence on lipid metabolism during develop-ment may also predispose the wax moth larvae to oxidative dam-age since polyunsaturated fatty acids are particularly susceptible to ROS attack and their metabolism can lead to lipid hydroperoxide formation.

There is no information on metabolism or biotransformation of aminoglycosides in insects. Midgut digestive enzymes such as glu-cosidase are likely important in metabolizing different types of gly-cosides in insects [53,54]. By cleaving some moieties from this aminoglycoside, metabolism of streptomycin by these enzymes may bioactivate the compounds, producing some reactive glyco-side metabolites as suggested by Hemming and Lindroth[54]for phenolic glycosides supplemented to artificial diets of another Le-pidopteran pest insect Lymantria dispar (L.).

Dietary streptomycin at highest concentration (1.0 g) resulted in significantly increased SOD activity concomitant with decreased CAT activity in the larval midgut tissue. This accords with the sug-gestion of Felton and Summers[24] that elevated levels of SOD activity can be accompanied by decreased CAT activities. In the present study, increased oxidative stress as evidenced by elevated MDA content might be associated with a depletion in SOD and CAT activities that are the main responses to dietary pro-oxidant expo-sure. These enzymes responsible for dismutation of superoxide radicals to H2O2and finally H2O2elimination, respectively, in

tis-sues[55]. Although, we have no direct evidence for generation of ROS by streptomycin, these may explain, at least, in part, the mas-sive production of superoxide radicals and H2O2and their

signifi-cant roles in streptomycin-induced inflammatory cellular responses in the midgut tissues. In supporting this suggestion, some antibiotics were shown to exert their bactericidal effects via the production of hydroxyl radicals in various animal models

[16]. However, not much attention has been given to related stud-ies in insects.

Alterations in the activity of glutathione-dependent enzymes, GST and GPx in the midgut of the larvae reared with some strepto-mycin concentrations could represent a way of preparing the in-sect for adaptive metabolic response to the elevation of lipid peroxidation as suggested by Miyamoto et al.[56]. We obtained similar adaptive responses, shown by increased activities of GST and GPx, against supplementation of inorganic insecticide, boric acid to G. mellonella diet[57]. GST is one of major phase II detoxi-fication enzymes conferring insecticide resistance in insects. It plays a vital role in prevention of oxidative damage by conjugating reactive species and by detoxifying lipid peroxidation products

[58]. In the present study, midgut GST activities were decreased in response to increased MDA content with a significant negative correlation. If this aminoglycoside antibiotic is metabolized to compounds (e.g., streptose, streptidine and methyl-L-glucosamine

as in mammalian system)[59] that cause oxidative stress, GST could afford protection against them. In a similar work with artifi-cial diet, GST activities in midgut of gypsy moth larvae were ele-vated in response to phenolic glycoside [54]. Our results are bolstered by the suggestion of Feng et al[60]that induced midgut GST activity of a pyralid moth, European corn borer, Ostrinia nubil-alis (L.) should enable the corn borer to metabolize chemicals more efficiently and therefore render the corn borer highly tolerant to the insecticides. GPx catalyzes the glutathione-dependent reduc-tion of lipid hydroperoxides and of hydrogen peroxide for detoxifi-cation. The increasing GPx activities attending decreasing GST activities at some streptomycin concentrations suggest that rear-rangement of separate activities of individual GST isoforms (GSTpx) may takes place during the antibiotic stress. Similar re-sults have been recorded for fat body and hemolymph of G. mello-nella larvae exposed to boric acid[57]as well as midgut tissue of another lepidopteran[61]under various environmental stressors. GSTpx against possible lipid peroxides may compensate for the low GPx activity for removal of H2O2and organic peroxides[30].

The present results show a significant depletion in midgut tis-sue level of ALT and AST, which are reliable biomarkers of tistis-sue in-jury, after oral treatment with streptomycin except for significant increase in AST with a dose level of 0.1 g antibiotic. This is

consis-tent with results of previous studies that, streptomycin-impaired transaminases activities shown in this study was associated with an increased oxidative stress [22,36]. These data suggest that streptomycin may cause metabolic dysfunction leading to tissue injury in the insect via generation of free radicals as in some verte-brate tissues on exposure to antibiotics [19]. This suggestion is confirmed that cytocidal effects of antibiotics were increased in a concentration-dependent manner by increasing concentrations of antibiotics[62]. ALT and AST activities are present in many tissues of insects[22,34]. These enzymes catalyze the reversible transfer of an amino group from amino acids to

a

[20]demonstrated that the mechanism of hydroxyl radical forma-tion induced by bactericidal antibiotics is the end product of an oxidatively damaged cellular death pathway involving the tricar-boxylic acid cycle, a depletion of NADH. A significant increase in the activity of the transaminase enzyme AST at some levels of streptomycin might be a result of a compensatory adaptive mech-anism of the larval midgut tissues for depletion in energy resources due to oxidative stress. This was in accordance with the findings of Eid et al.[33]dealing with the increased activities of the transam-inases as biochemical response in silk glands of P. ricini treated with some antibiotics.

In view of the data of the present study, it can be concluded that streptomycin-induced oxidative stress altering enzymatic antioxi-dant level and transaminases activities in the larval midgut tissues of G. mellonella. This raises the possibility that supplementation of streptomycin to diet are prone to form ROS in the oxidative midgut environment of the larvae. Together with our recent results[22], here we contribute further understanding of the mechanism underlying the action of antibiotics as a pro-oxidant on insect digestive physiology leading to deteriorated life parameters of G. mellonella.

Acknowledgments

I am grateful to Dr. David Stanley (USDA/ARS Biological Control of Insects Research Laboratory, Columbia, MO) for reading and pro-viding useful comments on a draft of this paper.

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