A R T I C L E
Ingestion of the anti-bacterial agent, gemifloxacin
mesylate, leads to increased gst activity and
peroxidation products in hemolymph of
Galleria
mellonella l. (lepidoptera: pyralidae)
Meltem Erdem
1Ceyhun Küçük
2Ender Büyükgüzel
2Kemal Büyükgüzel
31Ahmet Erdo ˘gan Vocational School of Health Services, Bülent Ecevit University, Zonguldak, Turkey
2Faculty of Science and Arts, Department of Molecular Biology and Genetics, Bülent Ecevit University, Zonguldak, Turkey
3Faculty of Science and Arts, Department of Biology, Bülent Ecevit University, Zonguldak, Turkey
Correspondence
Ceyhun Küçük, Faculty of Science and Arts, Department of Molecular Biology and Genetics, Bülent Ecevit University, 67100 Zonguldak, Turkey.
Email: cyhnkucuk@hotmail.com
Grant sponsor: Bülent Ecevit University Research Fund; Grant number: 2015-73769380-01.
Abstract
Gemifloxacin mesylate (GEM) is a synthetic, fourth-generation flu-oroquinolone antibacterial antibiotic that has a broad spectrum of activity against bacteria. GEM inhibits DNA synthesis by inhibiting DNA gyrase and topoisomerase IV activities. Recent research into insect nutrition and mass-rearing programs, in which antibiotics are incorporated into the culture media to maintain diet quality, raised a question of whether clinical antibiotics influence the health or biological performance of the insects that ingest these compounds. Because some antibiotics are pro-oxidant compounds, we addressed the question with experiments designed to assess the effects of GEM (mesylate salt) on oxidative stress indicators, using Galleria mellonella larvae. The insects were reared from first-instar larvae to adulthood on artificial diets amended with GEM at 0.001, 0.01, 0.1, or 1.0%. Feeding on the 1% diets led to significantly increased hemolymph contents of the lipid peroxidation product, malondialdehyde and pro-tein oxidation products, propro-tein carbonyl. All GEM concentrations led to increased hemolymph glutathione S-transferase activity. We inferred that although it was not directly lethal to G. mellonella larvae, dietary exposure to GEM exerts measurable oxidative damage, possi-bly on insects generally. Long-term, multigenerational effects remain unknown.
K E Y W O R D S
detoxification, Galleria mellonella, gemifloxacin, hemolymph, oxida-tive stress
1
I N T RO D U C T I O N
Antibiotics, including antibacterial, antifungal and antihelmintic agents, have been oncorporated into insect-rearing media for decades to determine their influence on life table parameters or their effective concentrations for control of microbial contamination in diets (Büyükgüzel & Yazgan, 2002; Singh & House, 1970). Antibacterials are classified according to their modes of action, that is, inhibitors of cell wall synthesis (e.g., penicillins), protein synthesis inhibitors (e.g., tetracyclines), and nucleic acid synthesis inhibitors (quinolones; Kohanski, Dwyer, & Collins, 2010). More recently, another, possibly common, antibacterial mechanism has been recognized. Antibiotics generate pro-oxidant hydroxyl radicals in Gram-positive and Gram-negative bacterial cells. In addition to the known drug–target interactions, these radicals kill bacteria by producing substantial oxidative damage to bacterial cell components (Kohanski, Dwyer, Hayet, Lawrence, & Collins, 2007). Although they are effective in killing pathogenic bacteria, antibiotics also exert vari-ous adverse side effects in humans, including serivari-ous ear and kidney damage. Kalghatgi et al. (2013) reported that clinically meaningful doses of some antibiotics lead to mitochondrial dysfunction and oxidative damage in patients, caused by reactive oxygen species (ROS). This finding opens the possibility that antibiotics incorporated into insect culture media may also exert damaging effects on insects consuming the laced media. Many insect species are rou-tinely maintained in very large colonies for research purposes and for large-scale insect pest management. The poten-tial influence of dietary antibiotics on insect biology is of importance because they may impose costs of maintaining insect cultures and potentially reduce the biological performance of insects reared for research and biological control programs.
For a specific example, the greater wax moth, Galleria mellonella, is maintained in many research programs. Antibi-otics have been used in larval diets to prevent microbial contamination in the diets and to obtain microorganism-free insects for research (Jarosz, 1981). We have developed G. mellonella as a model lepidopteran species to support our research into the influence of dietary pro-oxidant compounds, such as plant secondary metabolites, on insects and to investigate insect mechanisms responsible for attenuating oxidative damage. Büyükgüzel and Kalender (2007, 2009) conducted several studies with earlier antibiotics, including dietary penicillin, streptomycin, and fluconazole. These compounds led to the increased tissue concentrations of the lipid peroxidation product, malondialdehyde (MDA), an indicator of oxidative stress and also to increased activities of antioxidant enzymes including superoxide dismutase (SOD), catalase (CAT), glutathione S-transferase (GST), and glutathione peroxidase (GPx) in G. mellonella larval midguts. These antibiotics also influence life table parameters (Büyükgüzel & Kalender, 2008), slowing development and reduc-ing larval survival. We infer that reduc-ingestion of at least some antibiotics leads to negative effects on insects, registered as increased oxidative damage to cellular components.
Fluoroquinolones inhibit bacterial DNA replication by targeting DNA–topoisomerase complexes, which act in DNA strand breakage and rejoining (Kohanski et al., 2010). These antibiotics exert some antiproliferative and immunoregu-latory effects in experimental human cell lines, a primary human osteoblast cell line and two established lines, MG63 (osteosarcoma) and HeLa (derived from a cervical cancer) in addition to their antibacterial effects; others lead to cytotoxicity at high concentrations (Duewelhenke, Krut, & Eyse, 2007). Some first-generation DNA gyrase inhibitors, nalidixic acid and oxolinic acid, used in rearing the parasitoid, Pimpla turionellae, to control bacterial contamination, led to improvements in two biological performance parameters, increased survival rate and reduced developmental time when used in various combinations (Büyükgüzel, 2001). These inhibitors also reduce the cost for diet preser-vation without sacrificing the quality of the insects in mass-rearing facilities (Streett, Ni, & Lawrence, 2008). GEM (mesylate salt) is a recent fluoroquinolone antibiotic (launched in 2004; Hegde & Schmidt, 2005) with strong bac-tericidal activity. It is used for treatment of serious infections, including chronic bronchitis and multidrug resistant community acquired pneumonia. GEM is a fourth-generation DNA gyrase inhibitor, specifically targeting DNA gyrase (type II topoisomerase) and topoisomerase IV (Aldred, Kerns, & Osheroff, 2014). Based on the general actions of antibacterials as pro-oxidants, we posed the hypothesis that GEM exerts oxidative damage and stimulates increased GST activity in our model insect, G. mellonella. Here, we report on the outcomes of experiments designed to test our hypothesis.
2
M AT E R I A L S A N D M E T H O D S
2.1
Insect culture
Greater wax moth larvae and pupae were collected from infected hives in apicultural areas around Zonguldak, Turkey, and the newly emerged adults were used to maintain the stock culture. The insects were reared in 1,000 ml glass jars with an artificial diet (Bronskill, 1961) at 30± 1°C, 65 ± 5% relative humidity in constant dark-ness. The standard 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. Newly emerged adult females were placed in the jars and pro-vided a piece of old honeycomb on the diet for egg deposition and feeding of newly hatched larvae. Preparing and dispensing diets into containers to obtain eggs and transferring larvae onto diets followed our standard protocol. For continuing insect culture, these newly hatched larvae were reared through seventh instar at the same artificial diets. The last larval instars were transferred into new jars to obtain pupal and adult stages (Büyükgüzel, Hyršl, & Büyükgüzel, 2010).
2.2
Chemicals
All reagents were of analytical grade. Phenylmethylsulfonyl fluoride (PMSF), dithiotreitol (DTT), dipotassium hydro-gen phosphate (K2HPO4), potassium chloride (KCl), ethylendiaminetetraecetic acid (EDTA), butylated hydroxytoluene
(BHT), thiobabituric acid (TBA), trichloroacetic acid (TCA), bovine serum albumin (BSA), Folin-Ciolcalteu reagent, guanidine hydrochloride, 2,4-dinitrophenylhydrazine (DNPH), streptomycin sulfate, glutathione (GSH), 1-chloro-2,4-dinitrobenzene (CDNB), glycerol, ethanol, sodium chloride (NaCl), and phenylthiourea (PTU) were purchased from Sigma-Aldrich (St. Louis, MO). Gemifloxacin mesylate (GEM) was obtained from the Abdi ˙Ibrahim Medicine Co. (Istanbul).
2.3
Experimental designs
GEM was directly incorporated into diets at concentrations of 0.001, 0.01, 0.1, or 1.0%. Our preliminary experi-ments showed that these concentrations enable larvae to complete their adult development with gradually increas-ing mortality. Larvae reared on GEM-free diets were used as controls. Usincreas-ing standard laboratory rearincreas-ing conditions, we determined the influence of dietary GEM on lipid peroxidation and protein oxidation levels and activities of GST in hemolymph of seventh-instar larvae.
2.4
Biochemical assays
First-instar larvae were reared through seventh instars on an artificial diet amended with indicated GEM concentra-tions. The larvae were transferred into another jar lined with a filter paper for pupation. Last-instar larvae (of same chronological age, upon reaching seventh instars, 100–150 mg, n= 20 larvae/assay, four biologically independent replicates) were used for determining the MDA, PCO, and GST activity.
2.5
Sample preparation
The larvae were chilled on ice for 5 min and surface sterilized in 95% ethanol. Larval hemolymph was collected into cold Eppendorf tubes by amputating the second pair of prolegs. Cold homogenization buffer (w/v 1.15% KCl, 25 mM K2HPO4, 5 mM EDTA, 2 mM PMSF, 2 mM DTT, pH 7.4) and stored at−80°C. A few crystals of PTU were added to
each sample to prevent melanization. The cryotubes were kept at room temperature until the tissue began to thaw before using. Adults were homogenized at 4°C by an ultrasonic homogenizer (Bandelin Sonoplus, HD2070, Berlin, Germany) at 50 W, 40–50 s in homogenization buffer and subsequent centrifugation at 10,000× g for 10 min. The resulting cell-free extracts were collected for biochemical analysis. Protein concentrations were determined according
to Lowry, Rosebroug, Farr, and Randall (1951) using BSA as a quantitative standard. A dual beam spectrophotometer (Shimadzu 1700, UV/Vis) was used for all absorbance measurements.
2.6
Determination of mda, pco content, and gst activity
MDA content was assayed according to Jain and Levine (1995). MDA reacts with TBA to form a colored complex. MDA contents as an indicator of lipid peroxidation were determined after incubation for 45 min at 95°C with TBA (1% w/v). Absorbances were measured at 532 nm. MDA content were expressed as nanomoles per milligram protein, using 1.56× 105 M−1cm−1for extinction coefficient.
PCO content was assayed according to Levine, Williams, Stadtman, and Shacter (1994) with some modifications (Krishnan & Kodrík, 2006). PCO content was quantified spectrophotometrically after the reaction of carbonyl groups with DNPH leading to the formation of a stable 2,4-dinitrophenylhydrazone. Absorbances were measured at 370 nm. Results are expressed as nanomoles per milligram protein, using 22,000 M−1cm−1as the extinction coefficient. Pro-tein concentrations in guanidine solutions were measured spectrophotometrically (280 nm) and quantified with a BSA standard curve. PCO values were corrected for interfering substances by subtracting the A370/mg protein measured without DNPH.
GST (EC 2.5.1.18) activity was assayed by measuring the formation of the GSH/CDNB conjugate (Habig, Pabst, & Jakoby, 1974). The increase in absorbance was recorded at 340 nm for 3 min. The specific activity of GST was expressed as nanomole GSH–CDNB conjugate formed per minute per milligram protein using an extinction coefficient 9.6 mM−1cm−1. All assays were corrected for nonenzymatic reactions using corresponding substrate in phosphate buffer (50 mM, pH 7.0).
2.7
Statistical analysis
Data on the MDA and PCO contents and GST activity were analyzed by one-way analysis of variance (ANOVA). To determine significant differences between means, the least significant difference (LSD) test (SPSS, 1997) was used. When the F estimate exceeded the probability of 0.05, the differences were considered significant. Regression analysis was used to analyze possible correlations between dietary GEM concentrations and MDA, PCO concentrations, and GST activities (SPSS, 1997).
3
R E S U LT S
We recorded a significant increase in MDA in larvae exposed to the highest dietary GEM treatment (1.0%), although there was no correlation between GEM dose and MDA (Fig. 1). Higher PCO levels, about 91–99 nmol/mg protein, were obtained in larvae exposed to dietary GEM at 0.01, 0.1, and 1% (Fig. 2).
We detected GST activity as 1.32± 0.18 𝜇mol/mg protein/min in hemolymph of larvae reared on control diet. GST activity increased in an approximately linear manner, from about 3.8 mmol/mg protein/min to a high of about 6.3 nmol/mg protein/min in larvae exposed to dietary GEM at 0.001–0.1% of the diet. The highest GEM treatment did not result higher GST activity (Fig. 3), although the linear relationship between GEM dose and GST activity is significant (R2= 0.82, P < 0.05).
4
D I S C U S S I O N
The data reported in this article strongly support our hypothesis that GEM exerts oxidative damage and stimulates increased GST activity in our model insect, G. mellonella. The data are straightforward, showing that dietary GEM at 1% of the larval medium, led to increased MDA, a lipid peroxidation product, and at 0.01–1% level led to increased PCO
F I G U R E 1 The effects of GEM on hemolymph MDA content in G. mellonella larvae. Histogram bars depict mean amount of MDA (±SE; n = four biological replicates, 20 larvae per replicate) following larval development on diets amended with the indicated GEM dosage. Bars annotated with the same letter are not significantly different (P< 0.05, LSD test)
F I G U R E 2 The effect of GEM on hemolymph protein carbonyl content in G. mellonella larvae. Histogram bars depict mean amount of PCO (±SE; n = four biological replicates, 20 larvae per replicate) following larval development on diets amended with the indicated GEM dosage. Bars annotated with the same letter are not significantly different (P< 0.05, LSD test)
content, a protein peroxidation product. Finally, dietary GEM influenced hemolymph GST activity, which increased in a dose-related manner over the range of 0.001, 0.01, and 0.1% of the larval diets. Together, these points support our view that dietary GEM exerts oxidative damage and triggers increased activities of at least one anti-oxidative enzyme, GST.
Antibiotic action mechanisms, including gyrase inhibitors, on insect biological performance parameters are not understood. Dietary exposure to each of the three first-generation gyrase inhibitors, novobiocin, nalidixic acid, and
F I G U R E 3 The effect of GEM on hemolymph GST activity of G. mellonella larvae. Histogram bars depict mean GST activity (±SE; n = four biological replicates, 20 larvae per replicate) following larval development on diets amended with the indicated GEM dosage. Bars annotated with the same letter are not significantly different (P< 0.05, LSD test)
oxolinic acid, led to the slow development of the fifth instar, reduced survival of pupal, and adult stages in the endopar-asitoid Pimpa turionella (Büyükgüzel, 2001). However, when present in the diets in combination of two compounds, they led to an improved performance, registered as increased developmental rates and increased survival at pupal and adult stages (Büyükgüzel, 2001). We do not have directly comparable data on the influence of GEM on these perfor-mance parameters, however, our preliminary data show that dietary GEM leads to the increased biochemical measures of oxidative damage, considered as increased MDA and PCO contents. We predict that future studies will show that GEM exerts negative influences on insect biology.
More generally, based on their action mechanisms in microbes, it is not unreasonable to suggest that dietary GEM and other antibiotics can be deleterious to insects. There are sufficient data to support the view that many antibi-otics influence eukaryotic cells generally. For a single example, exposure to the bacteriocidal compounds, ciprofloxacin, ampicillin, or kanamycin, led to the increases in several indices of oxidative damage in mammalian cells, specifically, human mammary epithelial cells (MCF10A). Changes include increased ROS, mitochondrial superoxide, and hydrogen peroxide, and increased damage to DNA, proteins, and lipids. All three antibiotics also induced mitochondrial dysfunc-tion (Kalghatgi et al., 2013). Considered at the level of eukaryotic cell biology, cell funcdysfunc-tions known in mammals are also present in insect cells. Comparing insect hemocytes and mammalian neutrophils, both are responsible for lectin-mediated phagocytosis of invading bacterial cells, they have the same ROS, they both undergo degranulation, and they have similar cell-surface receptors, signaling pathways, and kinases (Browne, Heelan, & Kavanagh, 2013). Such broad similarities help to understand why at least some antibiotics can be harmful to human and insect cells. Some antifungal drugs, for a case in point, inhibit cytoskeleton development or sterol biosynthesis in mammals, and also impact west-ern tarnished plat bugs, Lygus hesperus (Alverson & Cohen, 2002). Rifampicin, an RNA synthesis inhibitor, significantly increased the time to adult emergence of the whitefly, Bemisia tabaci (Gennadius), and decreased the number of adults (Ruan, Xu, & Liu, 2006). It is not surprising that GEM can be damaging to mammalian and insect cells.
Several studies have confirmed the generation of oxidative stress indicators in G. mellonella hemolymph and midguts exposed to infectious agents and chemical pro-oxidants (Dubovskiy et al., 2008; Lozinskaya, Slepneva, Khramtsov, & Glupov, 2004). Here, we report that dietary GEM leads to increases in MDA and PCO in hemolymph and increased hemolymph GST activity. Hemolymph is an important tissue in these studies because hemolymph bathes all internal tissues and contains many circulating hemocytes. In effect, hemolymph is the natural environment of virtually all insect
tissues and cells and is responsible for many functions, including immunity (Büyükgüzel et al., 2010; Kanost, Jiang, & Yu, 2004).
The influence of GEM on hemolymph MDA and PCO concentrations operates in a threshold, rather than a dose– response pattern. Dietary GEM did not influence MDA concentrations in the range of 0–0.1% of the media, but led to a substantial increase at 1%. The effects of dietary GEM on PCO were similar, although at lower dietary concen-trations. There was no change in PCO at 0 and 0.001% dietary GEM, followed by large increases at 0.01, 0.1, and 1.0%. In general, dietary studies of pharmaceutical products in insects are limited by the lack of information on the pharmacokinetics—the times of product absorption, distribution, metabolism, and excretion—of these compounds in insects. A single report on the pharmacokinetics of the cyclooxygenase inhibitor, indomethacin (indo), in insect research illustrates the point. Indo is a nonsteroidal anti-inflammatory drug used to relieve pain and inflammation by block-ing prostaglandin biosynthesis at the cyclooxygenase step. Indo treatments were also used to investigate the roles of prostaglandins in insect physiology, although there was no information on the pharmacokinetics of the drug in insects. In later experiments, over 99% of injected radioactive indo was cleared from the hemolymph of tobocco hornworms,
Manduca sexta, in less than 3 min postinjection. The radioactivity was recovered from several tissues, including
hemo-cytes, integument, nerve cord, silk gland, gut epithelia, and fat body, mostly in the form of the injected indo. Most of the radioactive indo was excreted via frass over the first 14 h postinjection. A substantial proportion of the injected indo was converted into other products while in the frass (Miller & Stanley-Samuelson, 1996), presumably by micro-bial metabolism. More details of insect uptake, metabolism and excretion of indo (and many other compounds) are lacking. This is also true for GEM, MDA, and PCO. There are virtually no quantitative data on the formation, distribu-tion, metabolism, and excretion of these compounds in insects. This is a serious shortcoming in research designed to understand the biological impacts of dietary pro-oxidant compounds, including antibiotics and many plant secondary compounds, in insects.
AC K N O W L E D G M E N T S
This study was supported by Bulent Ecevit University Research Fund (project no.: 2015-73769380-01). We are grate-ful to Abdi ˙Ibrahim Medicine Company, Istanbul, Turkey, for providing gemifloxacin mesylate used in the study.
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