s2fieldsofstudy: Biology Medicine

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Modulatory effects of vagal stimulation on neurophysiological parameters and the cellular immune response in the rat brain during systemic inflammation

Background Stimulation of the vagus nerve has modulating, anti-inflammatory effects on the cellular immune response in the blood and the spleen, stabilizing brain function. Here, we aimed to investigate its potential effects on immune-to-brain communication focusing on neurophysiological readouts and leukocyte migration to the brain during severe sepsis-like endotoxemia. Methods Systemic inflammation was induced by intravenous administration of lipopolysaccharide (LPS; 5 mg/kg). Animals received either no manipulation of the vagus nerve, vagotomy, or vagotomy plus vagus nerve stimulation of the distal trunk. Somatosensory evoked potentials and evoked flow velocity response were measured for 4.5 h as indicators of brain function and neurovascular coupling, respectively. In addition, brain areas with (cortex) and without (hypothalamus) tight blood-brain barrier were studied separately using immunohistochemistry and RT-PCR. Moreover, plasma cytokine and leptin levels were analyzed by ELISA. Results LPS induced a decline of both neurophysiological parameters, which was prevented by vagus nerve stimulation. As for peripheral organs, LPS-stimulated neutrophil counts increased in the brain and colocalized in the brain with endothelial intercellular adhesion molecule (ICAM)-1. Interestingly, vagal stimulation reduced this colocalization and decreased nuclear translocation of the brain cell activation marker nuclear factor interleukin 6 (NF-IL6). Furthermore, it reduced the gene expression of inflammatory markers and extravasation signals (IL-6, CXCL-1, ICAM-1) in the hypothalamus but not the cortex linked to a moderate decrease in circulating cytokine levels (interleukin 6, tumor necrosis factor alpha) as well as lower plasma leptin concentration. Conclusions Our data suggest beneficial effects of anti-inflammatory vagus nerve stimulation on brain function by reducing the interaction of neurotrophil granulocytes with the brain endothelium as well as attenuating inflammatory responses in brain areas lacking a blood-brain barrier. Electronic supplementary material The online version of this article (doi:10.1186/s40635-016-0091-4) contains supplementary material, which is available to authorized users.

Background
Systemic inflammation results from host-related activation of innate immunity in response to infectious or non-infectious stimuli. In case of dysregulation of the inflammatory response, this naturally occurring process may bear deleterious effects for the host and frequently results in tissue injury, organ failure, and even mortality [1,2]. In recent years, many steps and key players in the pathway of immune activation have been described. It became evident that the initial systemic inflammatory response syndrome (SIRS) in general exceeds the needs for antimicrobial defense leading to the concept of host-related hyperinflammatory organ dysfunction as a relevant "motor" of developing sepsis [3]. Consequently, anti-inflammatory intervention strategies such as anti-cytokine antibody therapies have been developed, which interfere with the early steps of the inflammatory cascade. Whereas they were highly effective in experimental conditions, they failed clinically because manifest clinical symptoms in humans mostly occur at the later stages of the inflammatory response when the agents already lost their therapeutic relevance [4][5][6].
Nowadays, research concentrates on endogenous protection systems and aims to test their clinical feasibility. As such, vagus nerve stimulation is a prospective candidate for neuromodulation: several reports demonstrate a clinically relevant anti-inflammatory effect due to the stimulation of the efferent fibers of the vagus nerve. The efferent fibers originate from the nucleus (Ncl.) dorsalis vagi and represent the cranial parasympathetic autonomic nervous system innervating the heart, lung, and the oro-intestinal tract until the Cannon Böhm point at the left colon flexure. The efferent pathway of the anti-inflammatory vagus nerve reflex is still not fully understood but there are many reports showing a multisynaptically mediated functional influence on spleen lymphocytes involving the alpha7-nicotinic receptor [7]. However, the vagus also consists of afferent fibers terminating in the Ncl. tractus solitarii. The Ncl. tractus solitarius is linked to many sites in the brain affecting sickness behavior (several brain structures involved), temperature homeostasis (hypothalamus), the hormonal stress response (hypothalamus, pituitary), and the central sympathetic nervous system (Ncl. coeruleus) [8]. Furthermore, afferent fibers can induce cortical inflammation [9,10]. Due to the latter effect, most studies perform efferent vagus nerve stimulation by dissecting the nervus (N.) vagus and stimulating the distal part of the nerve trunk electrically [11].
Microcirculatory dysfunction is a key player of inflammation-related side effects leading to inappropriate blood supply and, therefore, functional failure of active organ cells. One of the most sensitive organs is the brain, which depends on continuous and appropriate blood supply because of its high energy demands and lack of stores for oxygen and/or energy substrates. Indeed, sepsis-related delirium is a frequent and early finding, which even precedes diagnosis of sepsis and appears in up to two thirds of sepsis patients [12].
In the present study, we aimed to extend our previous research on vagal antiinflammatory properties [13,14] to its effects on brain regions with (cortex) or without (hypothalamus) a tight blood-brain barrier. Therefore, several immune-to-brain signaling pathways (humoral and cellular) and different target structures related to the neurovascular coupling mechanism were studies in this well-established endotoxic lipopolysaccharide (LPS) sepsis model.

General animal preparation
Adult male Sprague Dawley rats (weighing 290 to 350 g) were purchased from Charles River (Sulzfeld, Germany). They were maintained on a 12-h light/dark cycle and housed four to a cage with food and water available ad libitum. Initially, rats were anesthetized with 1.5 to 3 % isoflurane, tracheotomized, paralyzed with pancuronium bromide (0.2 mg/kg/h; Inresa Arzneimittel GmbH, Freiburg, Germany) and mechanically ventilated (Harvard Rodent Ventilator; Harvard Apparatus, South Natick, MA, USA) with a 1:1 mixture of nitrogen and oxygen. For recording of blood pressure, blood sampling, and drug administration, the right femoral artery and vein were cannulated. Isoflurane anesthesia was then replaced by an intravenous application of an α-chloralose bolus (60 mg/kg; Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany) and maintained by a continuous administration of α-chloralose (40 mg/kg/h). A washout period of 60 min was maintained before the neurophysiological measurements were started. Arterial blood gas analysis and pH (blood gas analyzer model Rapidlab 348; Bayer Vital GmbH, Fernwald, Germany) as well as glucose (Glukometer Elite XL, Bayer Vital GmbH, Fernwald, Germany) and lactate (Arkray Inc. European Office, Düsseldorf, Germany) were measured at least every 60 min. To replace renal and perspirative fluid losses, a moderate volume therapy of 1.2 ml/h of 0.9 % NaCl was provided. Additionally, glucose concentrations in the blood were maintained at >60 mg/dl and body core temperature was maintained at 37°C using a heating pad.
All animal experiments were conducted in strict accordance with the National Institutes of Health Guide for Care and Use of Laboratory Animals and approved by the local ethics committee (ethics approval number GI 20/18-81/2011).

Neurovascular coupling measurement
The head of the animals was placed in a stereotaxic frame and the apex of the skull was exposed. To allow transcranial laser-Doppler flowmetry (LDF), the bone over the left parietal cortex was thinned with a saline-cooled drill (Dremel Moto-flex, Dremel Europe, Breda, The Netherlands) [15]. In accordance with the coordinates of the somatosensory cortex, the laser probe (BRL-100, Harvard Apparatus, Holliston, MA, USA) was placed 3.5 mm lateral and 1 mm rostral to bregma; this corresponds to the region of maximal hemodynamic response during contralateral forepaw stimulation [16][17][18]. For the continuous recording and processing of the laser-Doppler signal and mean arterial blood pressure, a data acquisition software (Neurodyn, HSE, March-Hugstetten, Germany) was used. Electric brain activity was measured monopolarily with one active calomel electrode about 0.5 mm behind the laser probe and one indifferent calomel electrode located on the nasal bone. Signals were recorded and amplified (BPA Module 675, HSE, March-Hugstetten, Germany) and in the following somatosensory evoked potentials (SEP) were averaged (Neurodyn acquisition software, HSE, March-Hugstetten, Germany). To calculate the SEP amplitudes, the difference between N2 and P1 was used.
To achieve somatosensory activation, rectangular bipolar pulses of 1.5 mA, 0.3 ms, and 2 Hz were applied by small needle electrodes placed under the skin of the right forepaw (PSM Module 676; HSE, March-Hugstetten, Germany). Stimulation with 1.5 mA ensured that there were no pain-associated changes in systemic blood pressure [16][17][18]. Electric stimulation was carried out for 30 s, followed by 30 s of non-stimulation. These activationrest cycles were performed ten times to increase the signal to noise ratio.
Whereas the Doppler does not measure absolute values, signal changes closely correlate with flow changes [16,17]. Therefore, flow velocity responses were averaged and the relative responses were calculated in relation to the resting phase, which was set to zero. The evoked flow velocity responses (EFVR) were then calculated from the averaged relative flow velocity signals under conditions of stimulation [13].

Vagus nerve stimulation
Both vagus nerves were exposed at the cervical level and carefully dissected from the common carotid artery in each rat. Animals received either no further manipulation of the vagus nerve (sham surgery (SHAM) and SHAM + LPS), a bilateral vagotomy (LPS + VGX), or a bilateral vagotomy and a stimulation of the distal trunk of the left nervus vagus (LPS + VGX + VNS) with a special nerve stimulation clamp (HSE, March-Hugstetten, Germany). For electrical stimulation, pulses of 2 mA, 0.3 ms, and 2 Hz were applied 15 min before administration of LPS and during the time course of the experiment and were only interrupted during the neurophysiological measurements [11].

Study design
Rats were assigned to the abovementioned groups in random order with 15 rats per group. Animals received 5 mg/kg body weight intravenous LPS (LPS from Escherichia coli, O111:B4; Sigma-Aldrich Chemie GmbH, Germany) resolved in 0.9 % NaCl or 0.5 ml of 0.9 % NaCl. LPS was administered slowly for 5 min. Experiments ended 4.5 h after LPS/vehicle administration, plasma samples were obtained, and brains were removed and stored at −80°C. The reason for limiting the study to 4.5 h after LPS application was that the blood-brain barrier and the macrocirculation were still in the normal range [19].

Tissue processing
Coronal 20 μm brain sections at the level of the subfornical organ (SFO, bregma −0.8 to −1.6 mm) were cut using a cryostat (2800 Grigocut E, Reichert-Jung, Nußloch, Germany). Brain sections were then thaw-mounted on poly-l-lysin-coated glass-slides and stored at −55°C for immunohistochemistry. Additionally, brain sections between the vascular organ of the lamina terminalis and the median eminence (bregma 0.6 to −2.8) were stacked on glass-slides, and the hypothalamus was dissected. The cortex and the hypothalamus were divided into two pieces each (left and right hemisphere) and separately stored at −55°C for polymerase chain reaction (PCR) analysis. Brain structures were identified using "The rat brain in stereotaxic coordinates" [20].
Specificity of the used antibodies has previously been tested [21]. Moreover, control experiments by substitution of the primary antibodies with non-immunized animals IgG were performed.

Microscopic analysis
A light/fluorescent Olympus BX50 microscope (Olympus Optical, Hamburg, Germany) and a black and white Spot Insight camera (Diagnostic Instruments, Visitron Systems, Puchheim, Germany) were used to acquire images from the stained sections. For each staining and time point, microphotographs were taken with the same exposure times using MetaMorph 7.7.5.0 software (Molecular Devices Inc., Downingtown, PA, USA). For NF-IL6 and the colocalization between neutrophil granulocytes and ICAM-1, the images were combined to RGB color images with help of the MetaMorph 5.05 software. All images were optimized for brightness and contrast using Adobe Photoshop 6.0 to the exact same extend within one analysis to preserve comparability (Adobe Systems Incorporated, San Jose, CA, USA).

Quantification
For the quantification of nuclear NF-IL6 immunoreactivity, the DAPI-stained nuclei were labeled with "1" and counted on microphotographs with a ×100 magnification. Subsequently, NF-IL6-positive nuclei were labeled with "2", counted, and given as percentage of all stained nuclei.
For the quantification of neutrophil granulocytes and ICAM-1, Fiji (Fiji Is Just ImageJ) software 2014 (open source software based on ImageJ modified by BioVoxxel, Mutterstadt, Germany) was used. After a median filter was applied to improve image noise, a threshold was set and the watershed function was used. Then, all particles over 20 pixels were automatically counted. For the number of neutrophil granulocytes, the number of particles on microphotographs with a ×5 and ×10 magnification and for the ICAM-1 immunoreactivity the percentage of the stained area on microphotographs with a ×20 magnification was used as readout.
The colocalization of neutrophil granulocytes and ICAM-1 was determined similar to the nuclear NF-IL6 immunoreactivity with a labeling of all neutrophil granulocytes and those that were colocalized with ICAM-1 on microphotographs with a ×10 magnification. The number of colocalized neutrophil granulocytes is given as percentage of all neutrophil granulocytes.
All images were processed in the same way, to guarantee comparability. For all quantifications, three sections of all animals and five animals of each group were used. The mean of the three sections was taken for calculating the mean of each group.
Brain maps for overviews were modified from the digital version of "The rat brain in stereotaxic coordinates" [20] using CorelDraw 9 (Corel Corporation, Ottawa, Canada).

Real-time PCR
Total ribonucleic acid (RNA) of the collected frozen cortex and hypothalamic sections (approximately 40 to 50 mg tissue) was extracted using Trizol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol. Reverse transcription of 1 μg total RNA was performed using 50 U murine leukemia virus reverse transcriptase, 50 μM random hexamers, and 10 mM deoxynucleoside triphosphates (dNTP) mix (Applied Biosystems, Foster City, CA, USA) in a total reaction volume of 20 μl. Afterwards, quantitative real-time PCR was performed in duplicate with preoptimized primer/probe mixture (TaqMan Gene Expression Assay, Applied Biosystems, Foster City, CA, USA) and TaqMan universal PCR Master Mix (Applied Biosystems, Foster City, CA, USA) on a StepOnePlus Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). The following cycling protocol was used: polymerase activation, 50°C for 2 min, denaturation, 95°C for 10 min, and 35 to 44 cycles of 15 s denaturation at 95°C followed by 1 min of annealing and elongation at 60°C.
The cDNA quantities were normalized by measurement of a housekeeping gene as a reference. Out of the six tested genes (Double-dye (Hydrolysis) probe geNorm 6 gene kit, ge-DD-6, Primer Design Ltd, Southampton UK), β actin (4352340E, Applied Biosystems, Foster City, CA, USA) was chosen as the best housekeeping gene using DataAssist 3.01 (Thermo Fisher Scientific Inc., Waltham, MA, USA) and NormFinder version 4 (Department of Molecular Medicine (MOMA) Aarhus University Hospital, Aarhus, Denmark).
Using the ΔΔCT method, sample values were calculated as x-fold difference from a control sample (SHAM, value determined as 1) within the same experiment. Samples from the same five animals of each group that were used for immunohistochemistry were also used for PCR analysis.
The detection limits for the assays were 31.3 pg TNFα/ml, 78 pg IL-6/ml, 15.6 pg IL-10/ml, and 0.2 ng leptin/ml, respectively. For TNFα, IL-6, and IL-10 measurements, 8 samples of the SHAM group and 13 samples of all septic groups were used, and for leptin, 5 samples of the SHAM group and 8 samples of all septic groups were used.

Statistics
Statistical analyses were performed using a one-way analysis of variance. In cases of significance, a Fisher post hoc test was applied (Statview, SAS, Cary, NA, USA). The SEP and EFVR data were analyzed separately for each time point. The significance level was set to P < 0.05. All data are presented as means ± standard error of the mean (SEM) or as means ± standard deviation (SD), respectively.

Clinical and neurophysiological results and systemic cytokine responses
Due to the α-chloralose-induced anesthesia, all animals developed a metabolic alkalosis and according to the volume administration a slight decrease in hematocrit at the end of the experiment. Using a 1:1 oxygen/nitrogen gas caused a supraphysiological partial pressure of oxygen in all groups while the partial pressure of carbon dioxide due to controlled ventilation remained in the physiological range. The glucose plasma concentration was kept in the physiological range during the 4.5 h of the experiment. Animals of the SHAM group showed stable glucose, lactate, blood gas, hemodynamic, and neurophysiological parameters during the time course of the experiment. LPS-treated animals developed a typical and reproducible severe sepsis syndrome: the blood pressure and pH decreased significantly, whereas the lactate plasma levels were induced. Septic animals also developed a decline of the SEP amplitudes and the evoked flow velocity responses. In line with previous reports [13], vagus nerve stimulation mitigated effects on blood pressure, pH, and lactate levels and stabilized neurophysiological data (Tables 1 and 2).
Regarding systemic cytokine levels, vagus nerve stimulation but not vagotomy significantly reduced TNFα and IL-6 levels in LPS-treated groups as compared to the SHAM + LPS group (Table 3).

Cytokine expression in the hypothalamus and cortex
Intravenous LPS application induced messenger RNA (mRNA) expression of TNFα, IL-1β, IL-6 and IL-10 in the cortex and hypothalamus of rats after 4.5 h (Fig. 1). Vagus nerve stimulation but not vagotomy significantly reduced the LPS-induced expression of IL-6 in the hypothalamus and cortex compared to the LPS + SHAM group. Vagal stimulation also decreased the IL-10 mRNA expression in the hypothalamus. Interestingly, vagotomy caused a significant reduction of the IL-1β mRNA expression in the cortex, whereas vagus nerve stimulation showed only a trend to a decline in the IL-1β mRNA expression (P = 0.07).

Effects on intracellular signal molecules of inflammation
LPS increased the expression of the inhibitor of kBα (IkBα), the suppressor of cytokine signaling (SOCS)3, and the nuclear factor interleukin (NF-IL)6 in the hypothalamus and the cortex (Fig. 2). In the hypothalamus, vagus nerve stimulation significantly inhibited the expression of SOCS3 and NF-IL6 mRNA expression as well as NF-IL6 immunoreactivity in the SFO and neighboring choroid plexus (Fig. 3). In the cortex, vagus nerve stimulation significantly attenuated the LPS-induced expression of IkBα and SOCS3 as well as NF-IL6. Interestingly, and only in the cortex, also, vagotomy blocked the increased expression of IkBα and SOCS3 as well as NF-IL6.
In addition, LPS induced the expression of the microglial activation marker CD68 in the hypothalamus and in the cortex. There were no differences in the CD68 expression between the septic groups in the hypothalamus. In the cortex, vagotomy lead to a reduced CD68 expression compared to the LPS + SHAM group.

Vagal effects on chemokines and adhesion molecules in the brain
LPS induced the expression of CXCL1 and the ICAM-1 in the hypothalamus and cortex on mRNA level. In the hypothalamus, vagus nerve stimulation significantly reduced the increased expression of CXCL1 and ICAM-1 (Fig. 5), whereas the immunohistological results of all groups did not show any difference between the constitutively increased levels in the SFO/choroid plexus (Additional file 1B-E). In the cortex, vagotomy and vagus nerve stimulation resulted in a reduced expression of the LPS-induced CXCL1 and ICAM-1 mRNA expression (Fig. 5), which did not result in immunohistologically significant differences between the LPS-treated groups (Additional file 1H-J). However, LPS induced an increase in ICAM-1 immunoreactivity compared to the sham group in particular in small brain blood vessels.
Vagus nerve stimulation reduces the number of neutrophil granulocytes, which are colocalized with ICAM-1 Immunohistochemical analysis revealed that LPS increased the number of neutrophil granulocytes in the SFO and neighboring choroid plexus as well as in the cortex (Fig. 6 and Additional file 2). However, there were no significant differences in the number of neutrophil granulocytes between the septic groups. Additionally, LPS increased the percentage of neutrophil granulocytes that were colocalized with ICAM-1 in the cortex. Interestingly, this effect was prevented by vagus nerve stimulation (Figs. 6c and 7).

Discussion
In our experiments, stimulation of the distal trunk of the vagus nerve showed antiinflammatory properties, as reported by several other authors [11,[22][23][24][25], reducing peripheral cytokines in the current study. Additionally, VGX + VNS was beneficial for brain microcirculation and brain function, preventing the LPS-induced decline of the SEP and EFVR. These results are in line with our previous work [13] and indicate that VNS might be a valuable tool for the treatment of sepsis and sepsis-associated delirium. Still, the underlying mechanisms, especially concerning the effects of VNS on the brain, are not well understood. Therefore, several transcription factors and cytokines were measured in a region with complete blood-brain barrier (BBB) (cortex) and a region lacking in parts a tight BBB (hypothalamus) as markers for central inflammatory activation [26]. Interestingly, VGX + VNS caused a significant decrease of the IL-6, IL 10, NF-IL6, SOCS3, CXCL1, ICAM-1, and COX2 mRNA expression in the hypothalamus indicating an anti-inflammatory effect of efferent VNS in this region. In contrast to the hypothalamus, vagotomy independent of VNS reduced the mRNA expression of IL-1β, IkBα, SOCS3, NF-IL6, CXCL1, ICAM-1, and COX2 in the cortex pointing to an antiinflammatory effect of VGX in the cortex. Here, we have to take into account the different properties of efferent and afferent vagus nerve stimulation as well as the different brain regions. While it was shown that efferent VNS has anti-inflammatory properties [11], afferent VNS signals can, for example, induce sickness behavior [27,28]. In line with our current results, Layé et al. [10] and Hansen et al. [9] have previously shown that subdiaphragmatic vagotomy blunted the LPS-or IL-1β-induced IL-1β induction in the hippocampus and hypothalamus independent of peripheral IL-1β expression. In this context, it has been suggested that the anti-inflammatory effect in the brain of an interruption of vagal afferents depends on the route of administration and the dose of the systemically injected inflammatory stimulus [8]. For example, Bluthe et al. reported that sickness behavior induced by intraperitoneal (ip) but not intravenous (iv) injections of IL-1β could be antagonized by vagotomy [29]. It was argued that the iv-route of administration caused a much stronger activation of the humoral afferent route of immuneto-brain signaling, which could override the pro-inflammatory effects of the afferent part of the vagus nerve. Others, however, provided evidence that fever induced by iv injections of LPS could be attenuated by vagotomy ( [8] for review). In an analogy, we found an anti-inflammatory effect of vagotomy in the brain independent from systemic effects of the vagal immune-modulation. One likely explanation might be that the BBB shields the cortex from a direct humoral or cellular inflammation from the blood (See figure on previous page.) Fig. 1 Real-time PCR analysis of cytokines in the hypothalamus (a, c, e, g) and in the cortex (b, d, f, h) 4.5 h after LPS or vehicle administration (n = 5 for each group). LPS induced an increased expression of TNFα (a, b), IL-1β (c, d), IL-6 (e, f), and IL-10 (g, h). Vagus nerve stimulation significantly reduced the increased expression of IL-6 in the hypothalamus and cortex compared to the LPS + SHAM group (P < 0.01) and the LPS + SHAM group (P < 0.001) and the LPS + VGX group (P < 0.05), respectively. Additionally, it reduced the IL-10 expression in the hypothalamus compared to the LPS + SHAM group (P < 0.05). However, it did not influence the TNFα or IL-1β expression. Vagotomy alone reduced the expression of IL-1β in the cortex compared to the LPS + Sham group (P < 0.05). Data are given as means ± SEM. Significance is given as: *compared to SHAM; *P < 0.05; **P < 0.01; ***P < 0.001; # compared as indicated; # P < 0.05; ## P < 0.01; ### P < 0.001; TNF tumor necrosis factor, IL interleukin, LPS lipopolysaccharide, SHAM sham surgery, VGX bilateral vagotomy, VGX + VNS bilateral vagotomy and distal vagus nerve stimulation stream. One prerequisite for this assumption is an intact BBB. In similar studies using the same animal model, we did not find hints for a breakdown of the BBB at the time point investigated [30]. In contrast, the hypothalamus contains regions that lack a tight BBB and, thus, are sensitive to direct systemic inflammatory factors. Systemic effects of anti-inflammatory vagus nerve stimulation, therefore, influence the local inflammatory response directly (Figs. 2 and 5). . LPS caused an increase of the analyzed mediators in the hypothalamus and cortex. In the hypothalamus, vagus nerve stimulation significantly reduced the SOCS3 expression compared to the LPS + SHAM group (P < 0.05) and the NF-IL6 expression compared to the LPS + SHAM group (P < 0.01) and the LPS + VGX group (P < 0.01). Interestingly, in the cortex, vagotomy alone, independent of vagus nerve stimulation, caused a highly significant decrease of the IkBα and SOCS3 as well as NF-IL6 (P < 0.001) expression. Data are given as means ± SEM. Significance is given as: * compared to SHAM; *P < 0.05; **P < 0.01; ***P < 0.001; # compared as indicated; # P < 0.05; ## P < 0.01; ### P < 0.001; IkBα inhibitor of kBα, SOCS suppressor of cytokine signaling, NF-IL6 nuclear factor IL-6, LPS lipopolysaccharide, SHAM sham surgery, VGX bilateral vagotomy, VGX + VNS bilateral vagotomy and distal vagus nerve stimulation Fig. 3 Immunohistochemical analysis of NF-IL6 in the SFO and neighboring choroid plexus (a-e) and in the cortex (f-j) 4.5 h after LPS or vehicle administration (n = 5). In the hypothalamus, there was no nuclear NF-IL6 immunoreactivity in the SHAM group (b) while all septic groups (c-e) showed strong intranuclear NF-IL6 signals. Vagus nerve stimulation (e) significantly reduced the percentage of nuclear NF-IL6 signals compared to the LPS + SHAM group (P < 0.01) and the LPS + VGX group (P < 0.01) (k). In the cortex, hardly any nuclear NF-IL6 signals could be detected after 4.5 h (g-j). The scale bar in picture B represents 25 μm and applies to pictures B-E and the scale bar in picture G represents 50 μm and applies to pictures g-j.
Cell nuclei are stained in blue and NF-IL6 in red. Data are given as means ± SEM. Significance is given as: * compared to SHAM; *P < 0.05; **P < 0.01; ***P < 0.001; # compared as indicated; # P < 0.05; ## P < 0.01; ### P < 0.001; NF-IL6 nuclear factor IL-6, SFO subfornical organ, LPS lipopolysaccharide, SHAM sham surgery, VGX bilateral vagotomy, VGX + VNS bilateral vagotomy and distal vagus nerve stimulation Interestingly, the cerebral mRNA expression of TNFα did not differ between the sepsis groups although the TNFα plasma concentration was reduced by VNS. This apparent contradiction was recently clarified by Sun et al. [30]: nicotine induced the expression of the microRNA (miR)-124 via α7 nicotinic acetylcholine receptors (α7nAChR) on macrophages. The miR-124 inhibits the tumor necrosis factor-alpha  -1 (b, d), and the microglial activation marker CD68 in the hypothalamus (a, c, e) and in the cortex (b, d, f) (n = 5). COX2 and mPGES-1 expression was induced 4.5 h after LPS administration in the cortex as well as in the hypothalamus. The mPGES-1 expression did not differ significantly between the septic groups. In the hypothalamus, vagotomy increased and vagus nerve stimulation decreased COX2 expression resulting in significant differences (P < 0.05) whereas in the cortex both interventions resulted in a significant decrease in expression (both P < 0.05). Moreover, LPS induced the expression of CD68 in the hypothalamus and cortex. While there were no differences between the septic groups in the hypothalamus, vagotomy leads to a reduced CD68 expression in the cortex compared to the LPS + SHAM group (P < 0.01). Data are given as means ± SEM. Significance is given as: * compared to SHAM; *P < 0.05; **P < 0.01; ***P < 0.001; # compared as indicated; # P < 0.05; ## P < 0.01; COX cyclooxygenase, mPGES microsomal prostaglandin synthase, LPS lipopolysaccharide, SHAM sham surgery, VGX bilateral vagotomy, VGX + VNS bilateral vagotomy and distal vagus nerve stimulation converting enzyme (TACE) causing a reduced concentration of TNFα despite of an increased TNFα mRNA expression. It is known that microcirculatory failure is a motor of organ dysfunction and microcirculatory changes precede the decline of brain function [31]. As VNS protects brain  CXCL1 (a, b) and ICAM-1 (c, d) in the hypothalamus (a, c) and cortex (b, d) as well as the quantification of immunohistochemical analysis of ICAM-1 in the SFO and neighboring choroid plexus (e) and in the cortex (f) (n = 5). CXCL1 and ICAM-1 expression was induced 4.5 h after LPS administration. In the hypothalamus, vagus nerve stimulation leads to a decreased expression of CXCL1 compared to the LPS + SHAM group (P < 0.01) and a decreased expression of ICAM-1 compared to the LPS + SHAM group (P < 0.01) and the LPS + VGX group (P < 0.05). The immunohistochemical analysis of the SFO and neighboring choroid plexus revealed no differences between the different groups. In contrast to the hypothalamus, in the cortex, vagotomy independent of vagus nerve stimulation caused a decreased expression of CXCL1 (P < 0.05 and P < 0.01) and ICAM-1 (P < 0.001) compared to the LPS + SHAM group, although this did not result in immunohistologically significant differences. Data are given as means ± SEM. Significance is given as: * compared to SHAM; *P < 0.05; **P < 0.01; ***P < 0.001; # compared as indicated; # P < 0.05; ## P < 0.01; ### P < 0.001; ICAM intercellular adhesion molecule, SFO subfornical organ, LPS lipopolysaccharide, SHAM sham surgery, VGX bilateral vagotomy, VGX + VNS bilateral vagotomy and distal vagus nerve stimulation microcirculation, it seems likely that VNS may interfere with some mechanism regulating brain blood flow. One key factor in the regulation of cerebral blood flow is nitric oxide (NO). However, previous work showed that VNS does not influence the nitric oxide system in the brain [13] under septic conditions. Other important factors are prostaglandins which are linked to inflammation as well as regulation of the cerebral blood flow [32,33]. In the literature, the influence of nicotinergic stimulation on the synthesis of PGE2 is controversially discussed. Stimulation of monocytes and microglia with nicotine caused in some studies an induction [34][35][36] of PGE2 while it had no influence [37,38] or even lead to an inhibition [38,39] of PGE2 synthesis in others. However, Le Maitre et al. [37] showed that PGE2 seems to influence the antiinflammatory effects of VNS. In their study, mPGES-1 knockout animals were not able to develop a rise of acetylcholine in the spleen and a reduction of pro-inflammatory cytokines after VNS. Nevertheless, they were not able to detect an influence of VNS on the PGE2 expression in the periphery or the brain, so that the significance of PGE2 for the anti-inflammatory actions of VNS remained unclear. However, results of our current study point to an influence of VNS on PGE2 synthesis in the hypothalamus. Further analysis of arachidonic acid derivates could clarify this objective.
In addition to humoral factors, leukocytes are also thought to contribute to the pathogenesis of organ dysfunction during sepsis. While VNS did not influence microglial activation, Fig. 6 Immunohistochemical analysis of the number of neutrophil granulocytes in the SFO and neighboring choroid plexus (a) and the cortex (b) and their colocalization with ICAM-1 (c) 4.5 h after LPS or vehicle administration (n = 5). LPS increased the number of neutrophil granulocytes in the SFO and neighboring choroid plexus and the cortex as well as their colocalization with ICAM-1. There were no differences in the number of neutrophil granulocytes between the septic groups, but interestingly, vagus nerve stimulation reduced the percentage of neutrophil granulocytes that were colocalized with ICAM-1 compared to the LPS + SHAM group (P < 0.01) and the LPS + VGX group (P < 0.01). Data are given as means ± SEM. Significance is given as: * compared to SHAM; *P < 0.05; **P < 0.01; ***P < 0.001; # compared as indicated; # P < 0.05; ## P < 0.01; ### P < 0.001; ICAM intercellular adhesion molecule, SFO subfornical organ, ChP choroid plexus, LPS lipopolysaccharide, NG neutrophil granulocytes, SHAM sham surgery, VGX bilateral vagotomy, VGX + VNS bilateral vagotomy and distal vagus nerve stimulation our results suggest a modulatory effect of VNS on the migration of neutrophil granulocytes to the brain. In this regard, VNS reduced the mRNA expression of the neutrophil-specific chemokine CXCL1 and the adhesion molecule ICAM-1 in the hypothalamus, indicating a VNS-induced reduction of migration signals for neutrophil granulocytes. Interestingly, and possibly due to the early time point of investigation, protein-level immunohistochemical analysis did not show any differences of ICAM-1 between the septic groups neither in the SFO and neighboring choroid plexus nor in the cortex. This might be due to different regulatory processes on mRNA and protein level, which act in a time-dependent manner. Neutrophil granulocytes are thought to migrate into the brain parenchyma 12 to 24 h after an insult [40][41][42][43][44][45]. At the early time point (4.5 h) of this study, they were probably still adhering to and rolling on endothelial cells [45]. Interestingly, the number of neutrophil granulocytes counted in the cortex did not differ between the septic groups, but we could show here, for the first time, that VNS reduced the colocalization between ICAM-1 and neutrophil granulocytes indicating a decreased interaction and adhesion of neutrophil granulocytes and ICAM-1 on cerebral blood vessels. This might contribute to the beneficial effects of VNS on brain microcirculation. As ICAM-1 protein expression was not influenced by VNS, this might rather point to a VNS-induced modulation of neutrophil granulocytes themselves. Mechanism by which VNS modulates neutrophil granulocytes should be further analyzed in future studies. As one possible mechanism, we analyzed leptin plasma concentration, which was reduced by VNS. Leptin is well known as appetite-regulating hormone but recently its function as neuro-immune mediator became known [46]. In this regard, leptin is involved in the migration of neutrophil granulocytes to the brain [40,44,47]. Therefore, upon other in part mentioned mechanisms, VNS might also inhibit neutrophil migration to the brain in a leptin-dependent manner.
Another hint for the relevance of the cerebral microcirculation on neuronal function in the early inflammation stages is that the cortical anti-inflammatory effects of vagotomy neither protected from neurovascular dysfunction nor decline in evoked potential amplitudes. The matter of vagus nerve stimulation on the intact nerve, which most likely resembles clinical conditions using external nerve stimulators might be addressed in future studies. In this context, the possibly opposing effect of transcutaneous (clinical) stimulation of the intact vagus nerve on systemic inflammation and inflammatory modifications within the brain should be considered.

Conclusions
We addressed for the first time the potential brain-related mechanisms of the protective effect of efferent vagus nerve stimulation on neurophysiological parameters, e.g., neurovascular coupling during an early phase of severe inflammation. Contributing factors pertain to (1) reduced interaction of neutrophil granulocytes with brain endothelial cells linked to reduced circulating leptin levels and (2) an attenuated inflammatory response of the brain as reflected by changes in several inflammatory markers including inflammatory transcriptions factors namely NF-IL6. (3) Moreover, new insights were revealed into the brain area-specific influences of vagus nerve stimulation/vagotomy depending on its access to circulating mediators, e.g., cortical areas compared to hypothalamic structures in close vicinity to circumventricular organs with a leaky blood-brain barrier.


Matrix metalloproteinase-9 Gene-1562C>T Gene Polymorphism and Coronary Artery Disease in the Chinese Han Population: A Meta-Analysis of 5468 Subjects

Background: Multiple studies indicate that the matrix metalloproteinase-9 (MMP-9)-1562C>T gene polymorphism may be associated with an increased risk of coronary artery disease (CAD) in the Chinese Han population. However, a clear consensus has yet to be established. Objective and methods: A meta-analysis of 5468 subjects from 10 separate studies was performed to explore the possible relationship between the MMP-9-1562C>T gene polymorphism and CAD within the Chinese Han population. Pooled odds ratio (ORs) for the association and the corresponding 95% confidence intervals (CIs) were evaluated by a random or fixed-effect model. Results: Our analysis confirms the association between the MMP-9-1562C>T gene polymorphism and an increased risk of CAD within the Chinese Han population under allelic (OR: 1.60, 95% CI: 1.25–2.04, P = 0.0002), recessive (OR: 3.05, 95% CI: 1.67–5.56, P = 0.0003), dominant (OR: 2.23, 95% CI: 1.49–3.35, P = 0.0001), homozygous (OR: 3.41, 95% CI: 1.87–6.23, P < 0.0001), heterozygous (OR: 2.03, 95% CI: 1.40–2.93, P = 0.0002), and additive genetic models (OR: 1.78, 95% CI: 1.33–2.39, P < 0.0001). Conclusions: In the Chinese Han population, the MMP-9-1562C>T gene polymorphism is correlated with an increased risk of CAD. Therefore, Han Chinese carriers of the -1562T allele may be at an increased risk of CAD.

INTRODUCTION
Coronary artery disease (CAD) is a chronic condition with both hereditary and environmental factors. The increasing prevalence of unhealthy lifestyles in China (i.e., extended periods of sitting, a more sedentary lifestyle, a high-fat diet), the general aging of the Chinese population, and the increasing rates of hypertension and diabetes have damaged the quality of life of many Chinese people and sharply increased CAD-associated morbidity and mortality . However, recent advancements in molecular biology have allowed researchers to better elucidate the pathogenic mechanism of CAD.
Matrix metalloproteinases (MMPs), a large family of zincdependent proteolytic enzymes that have special functions in extracellular matrix (ECM) degradation have been a particular rich area of research. MMPs play a role in multiple pathophysiological processes such as ECM degradation, inflammation response, tumor metastasis, and atherosclerosis (Castro and Tanus-Santos, 2013). Enhanced MMP expression and activity also play a pivotal role in early arteriosclerosis, plaque rupture, myocardial infarction, and heart failure (Tanner et al., 2011). MMP-9 (also known as gelatinase B) is one the most extensively researched MMPs and exhibits enhanced expression in atherosclerosis injury sites. It has been also found that there were many gene sequence variations in MMP-9 gene, of which the locus rs3918242 (-1562C>T) in the promoter region was the most reported gene locus.
MMP-9 gene, located in 20q11.2-13.1, spans 7.7 kb and contains 13 exons. MMP-9-1562C>T mutation occurs in a substitution of cytosine (C) for thymine (T). This mutation may increase MMP-9 gene expression and risk of CAD development by reducing the binding affinity with transcription inhibition proteins.
Although many studies on the association between MMP-9-1562C>T gene polymorphism and CAD have been performed in China, the composite of these studies fail to provide a consensus. In 2005, Tang et al found that MMP-9-1562C>T gene polymorphism was significantly associated with CAD in a Zhejiang population and T allele increased the CAD risk (Tang et al., 2005). In 2007, Chen et al also reported a similar result in another Hunan population (Chen et al., 2007). In contrast, Wu et al failed to find an association between MMP-9-1562C>T gene polymorphism and CAD in a Beijing population in 2009 (Wu et al., 2009) and Zhi et al observed no significant effects for MMP-9-1562C>T gene polymorphism on CAD risk in a Jiangsu population (Zhi et al., 2010).
We performed the current meta-analysis with the hopes to provide a valuable conclusion on the association between MMP-9-1562C>T gene polymorphism and CAD in the Chinese Han population.

Publication Search and Inclusion Criteria
The Web of Science, PubMed, Embase, the China Biological Medicine Database, and the China National Knowledge Infrastructure electronic databases were searched using the terms "matrix metalloproteinase-9," "coronary artery disease, " or "coronary heart disease, " and "polymorphism" in our initial search. Retrieved studies were published between 2005 and 2010 with the last study updated on March 26, 2016.
To meet our inclusion criteria, studies had to (a) evaluate the association between MMP-9-1562C>T gene polymorphism and CAD in the Chinese Han population. (b) diagnose CAD according to the clinical symptoms combined with examination results (i.e., coronary arteriography, electrocardiogram, treadmill exercise test, echocardiography, myocardial perfusion imaging by Emission Computed Tomography, etc.) with a minimal stenosis rate of the major coronary artery diameter of more than 50% (c) be a case-control or cohort study published in an official journals or as a postgraduate dissertation.

Data Extraction
Data was extracted according to a standardized protocol. Two investigators searched for duplicates while a third served as an arbiter to resolve possible disagreements. Duplicate papers, those that violated the inclusion criteria, or those that provided deficient data were removed. Identical data sets used in different studies by the same authors were used once. Abstracted data consisted of the following items: the first author's name, publication year, region, number of genotypes, genotyping method, study design, age, gender, and total number of cases and controls.

Statistical Analysis
The odds ratio (OR) and its corresponding to 95% confidence interval (CI) were used to measure the association between MMP-9-1562C>T gene polymorphism and CAD. The Chisquare-based Q-test was adopted to measure the effects betweenstudies heterogeneity (P < 0.05 level) (Cochran, 1968).Variation due to heterogeneity was assessed by the inconsistency index I 2 . If heterogeneity were present in the study, the randomeffects model would be used to assess the combined OR (the DerSimonian and Laird method) (DerSimonian and Laird, 1986). Otherwise, the fixed-effects model would be used (the Mantel-Haenszel method) (Mantel and Haenszel, 1959). The pooled OR was determined by Z-test and significance was set at P < 0.05.
The Fisher's exact test was used to evaluate the Hardy-Weinberg equilibrium (HWE) (P < 0.05). Potential publication bias was assessed by Egger ′ s linear regression test on the natural log scale of the OR to detect funnel plot asymmetry (P < 0.05 level) (Egger et al., 1997). The statistical analysis was performed by STATA 11.0 software (StataCorp, College Station, TX).

Studies and Populations
Ten of the nineteen retrieved papers fit the inclusion criteria. Of the nine excluded studies, one was a duplicate, four were reviews, and another four were irrelevant to our interests. No study was excluded for deviation from HWE. The ten studies compiled the data from 3168CAD patients and 2300 controls ( Table 1, Presentation 1 in Supplementary Material; Tang et al., 2005;Meng et al., 2006;Chen et al., 2007;Wang et al., 2007;Wu et al., 2009;Gao and Wang, 2010;Ma et al., 2010;Yong and Shi, 2010;Zhang et al., 2010;Zhi et al., 2010) and represented seven provinces (Shanxi, Xinjiang, Jiangsu, Zhejiang, Hunan, Tianjin, and Beijing). All subjects were of Han ethnicity. However, there were substantial differences in the size of the total patient population between these studies. The Wu N study alone contributed 1356 CAD patients and 689 controls for this  meta-analysis. Differences in patient number may be factor in the lack of consensus on this topic.

Combined Analyses
There was a significant association between MMP-9-1562C>T gene polymorphism and CAD in the Chinese Han population under allelic  Figures 1-6).
Under the allelic genetic model, CC sample size of CAD group (CC1, P = 0.006), CT sample size of CAD group (CT1, P = 0.005), TT sample size of CAD group (TT1, P = 0.010), CC sample size of control group (CC0, P = 0.023), total sample size of control group (T0, P = 0.034), and weight (%) (P = 0.030)were possible confounding factors that could partially explain heterogeneity between studies.
Under the additive genetic model, CC1 (P = 0.013), CT1 (P = 0.012), and TT1 (P = 0.016) were the confounding factors. In the allelic and additive genetic models, CT1 plays a central role  in explaining the source of heterogeneity. According to CT1, the whole population was separated into two subgroups. The studies with CT1 < 30 were grouped into subgroup 1 and the residual studies with CT1 ≥ 60 belonged to subgroup 2.

Sensitivity Analysis
In the current meta-analysis, the sensitivity analysis was performed. After the Wu et al. was removed from the current meta-analysis, the association between MMP-9-1562C>T gene polymorphism and CAD was further strengthened under the allelic genetic model (OR: 1.72, 95% CI: 1.35-2.19, P = 1.3 × 10 −5 , P heterogeneity = 0.04, I 2 = 50.0%; Wu et al., 2009). Removal of other studies respectively from the current studies did not change the results from the original analysis. Hence, the Wu et al study should be the high sensitivity study in the current meta-analysis.

Bias Diagnostics
The publication bias of the studies was evaluated by funnel plot and Egger's test. There was no visual publication bias in the funnel plot (Figure 7). No statistically significant difference was detected in the Egger's test, implying no publication bias existed in the current meta-analysis under the recessive genetic model (T = −0.73, P = 0.487).

DISCUSSION
In the present meta-analysis, we found a significant association between MMP-9-1562C>T gene polymorphism and CAD in the Chinese Han population under allelic (OR: 1.60), recessive (OR: 3.05), dominant (OR: 2.23), homozygous (OR: 3.41), heterozygous (OR: 2.03), and additive genetic models (OR: 1.78). Hence, it has been concluded that in Chinese Han population, the MMP-9-1562C>T gene polymorphism may be associated with the increased CAD susceptibility among Han Chinese.
What contributed to the recent controversy over the association between MMP-9-1562C>T gene polymorphism and CAD? The meta-regression used to reveal the source of the heterogeneity detected under allelic, dominant, heterozygous and additive genetic models (P heterogeneity < 0.05) suggests that patient number may have been the confounding factor. In the heterogeneity source analysis, CT1 was possibly indicated to be the main heterogeneity source under allelic and additive genetic models. Although the subgroup analysis stratified by CT1 showed a significantly increased risk of CAD in both subgroups, only one subgroup exhibited heterogeneity. Hence, CT1 was the main confounding factor contributing to the heterogeneity under the allelic and additive models. Similarly, T1 and CC0 were major confounding factors under heterozygous and dominant models, respectively. CT1, T1, CC0 may be better matched between the CAD and control groups under these genetic models.
Sensitivity analysis showed that our meta-analysis was most sensitive to the Wu et al study. Although the pooled analysis result without Wu et al study was different from the original result (Wu et al., 2009), they are still consistent in associating the presence of the gene polymorphism with an increased risk of CAD.
MMPs belong to a neutral protease family that contains zinc ions. MMP-9 is the leading MMP expressed and secreted by the vascular cell walls. It is also secreted by monocytes, neutrophils, vascular smooth muscle cells (VSMCs), and endothelial cells. The relative molecular weight of MMP-9 is 92KD. In its active form its molecular weight is 84KD. MMP-9 can degrade extensive ECM substrates, including Type IV collagen, which plays a key role in the revascularization, inflammation response, and atherosclerosis progression. Research has shown an increased MMP-9 expression level in the atherosclerotic arteries of human and animals compared to normal arteries. The MMP-9 degradation activity was most located in the shoulder regions of plaque, the lipid core margin, and micro-vessels formation regions. This suggests that MMP-9 may be associated with the coronary artery   plaque stability and myocardial infarction (Speidl et al., 2011).
The animal experiments have discovered that the atherosclerosis lesions and VSMCs intima migration in the MMP-9 gene knock-out mice were remarkably decreased than that in the wild-type mice (Ye, 2006). The clinical researches have confirmed that the high MMP-9 expression level was correlated with the premature CAD, unstability of the coronary atherosclerosis plaque, the in-stent restenosis and arterial aneurysm formation (Jones et al., 2006). The prospective researches have shown that plasma MMP-9 contents can serve as the prediction indicator for the cardiovascular diseases mortality (Blankenberg et al., 2003).
MM9 is regulated primarily at the transcriptional level. The MMP-9-1562C>T gene polymorphism is located in crucial regulatory elements, including the 9 bp sequence GCGCAC/TGCC (−1567→−1559), a potential binding site for transcription inhibition proteins (Zhang et al., 1999). In 2002, Cho et al reported a change in bond zone structure and a weakened binding capacity between DNA and transcription inhibition protein, when the MMP-9-1562C allele was replaced by -1562T allele (Cho et al., 2002). This generates a high and low activity promoter genotype (CT/TT and CC, respectively) with increased transcription with the high activity promoter genotypes.
Increased MMP-9 expression may contribute CAD development through a number of pathways. It may promote the VSMCs proliferation and migration, promote the injured vascular remodeling, and/or promote the plaque rupture and lead to the thrombosis, resulting in the acute coronary syndrome known as myocardial infarction (Galis et al., 2002).
Past meta-analyses on the association of MMP-9-1562C>T gene polymorphism and CAD (Li et al., , 2013Niu and Qi, 2012) show weaknesses in their method. Although the distribution of genes differs between populations, these metaanalyses mixed ethnic Han Chinese with other ethnicities. The present meta-analysis, on the other hand, studies only the Han Chinese population. In addition, their initial search for manuscripts was not as comprehensive as that of the current meta-analysis, making their work less objective and credible.
However, this meta-analysis is not without limitations. The large-scale studies present in the analysis were not adequate to fully elucidate the complex relationship between the MMP-9-1562C>T gene polymorphism and CAD. The CAD susceptibility is also influenced by environmental factors, such as smoking, diabetes, dyslipidaemia, air pollution, inflammation, and psychological factors Agüero et al., 2013;Parruti et al., 2013;Wichmann et al., 2013). It is quite possible that the MMP-9-1562C>T gene polymorphism interact with a risk factor that was not within the scope of this study. There are also many other MMP-9 gene polymorphisms as P574R, R+279Q, and R668Q that influence the MMP-9 serum level (Zhi et al., 2010).
In conclusion, the current meta-analysis indicates that the MMP-9-1562T allele may increase the CAD risk among the Chinese population. This result has the potential to guide the therapy strategy for a CAD patient. Taking into account the limitations mentioned above, it remains necessary for these results to be verified by future studies.

AUTHOR CONTRIBUTIONS
YL researched data. YL and CZ wrote manuscript, researched data. YL, YZ, XY, HK, and GG reviewed/edited manuscript. YL, HG, and YQ contributed to discussion, reviewed/edited manuscript. YL, JW, and XW researched data, contributed discussion.


s2fieldsofstudy: Biology Chemistry

text:
ANTICANCER ACTIVITY OF SILVER NANOBIOCONJUGATES SYNTHESISED FROM PIPER BETLE LEAVES EXTRACT AND ITS ACTIVE COMPOUND EUGENOL

Objective: Our present study deals with the anticancer property of silver nano bio conjugates synthesized from the methanolic extract of Piper betle leaves and the pure compound eugenol. Methods: The anticancer activity of the nano bio conjugates synthesized from the extract and pure compound, in comparison with their respective non-conjugated raw material, were studied using cancer cell lines. The anticancer activity was analyzed by cell viability and staining techniques. The cell viability was determined by the MTT assay and the apoptosis was evaluated by the AO/EtBr staining method, which was carried out in both the cancerous (oral carcinoma (KB) cell lines) and non-cancerous cells (primary cultured human buccal cells). The phase of cell death was assessed by the flow cytometry followed by the cytotoxicity assessment. Results: The results revealed that Piper betle leaves as well as their active phenolic compound eugenol exhibited strong anticancer activity in KB oral carcinoma cells. The anticancer effect of the extract was attributable to the component phenolics, eugenol respectively in betel leaves, as the phenolics also exhibited strong cytotoxicity in cancer cells. The anticancer activity increased by a marked magnitude when the extract/compound was administered as nano-bio conjugates. The significant observation was the differential effect evoked by the AgNPs, which was non-toxic to noncancerous buccal cells while evoking a strong cytotoxicity in the cancer cells. Conclusion: The results proved that the phytoconstituents like phenols in the betel leaves play a major role in the reduction of nano bio conjugates. The anticancer properties of silver nano bio conjugates were higher in the conjugated nanoform than the non-conjugated form.

INTRODUCTION
Currently about 50% of cancers in men and 20% of cancers in women are related to tobacco use, which has led to oral cancer death in the age group of 35-64 y. Developing countries of the South Asian region have a higher incidence of oral cancer compared to developed countries [1,2]. The standard treatment followed all over the world is radiation therapy, or a combination of radiation therapy, chemotherapy and surgical removal of cancer. In spite of such interventions, cancer continues to take lives. Researchers are also intensifying their efforts on various therapy and trails on immunotherapy, antiangiogenic therapy, gene and viral therapy, cancer stem cell therapy and targeted therapy [3].
Over the last few decades, about 85,000 compounds have been screened for cancers, but only a few could be considered as effective and promising anti-cancer drugs [4]. Recently, the field of cancer therapies has been improved by many diverse scientific disciplines in order to fight better against cancer. Nanotechnology, which involves the creation, manipulation, and application of structures in the nanometer size range, has started revolutionizing cancer diagnosis and therapy [5]. Nanomedicine represents an innovative and multidisciplinary field that exploits nanotechnology to utilize in disease diagnosis and treatment. The main feature of these nanosystems is that their surfaces can be functionalized, exploiting reactive terminal groups, with specific proteins, peptides or monoclonal antibodies that are able selectively to bind a site of action or a particular target tissue, without interacting with other cells. They also improve the stability of drugs and release the drug in a constant, uniform concentration and in a controlled system, leading to reduced side effects [6]. Nowadays nanotechnology-based approaches to overcome the problem occur in the conventional system for treatment and the diagnosis of cancer [7].
The green production of nanoparticles using naturally occurring agents such as plant extract, microorganisms and biodegradable sources like reductant and capping agents, have made a remarkable attraction toward nanotechnology. Piper betle (betel leaves) have been used in our Indian traditional medicine system for digestive and pancreatic lipase stimulant activities [8]. The leaves of Piper betle have been employed in traditional herbal remedies. Active phenolic compounds such as chavibetol, piperol, piperbetol, hydroxychavicol and eugenol are enriched in the betel leaves, among which eugenol is major phenolic possess remarkable chemotherapeutic and chemopreventive potential, and it is inexpensive and non-toxic [9][10][11].
Based on the preliminary study conducted in our laboratory, the method of synthesis of silver nanobioconjugates was optimized using the methanolic extracts of Piper betle leaves as well as their active phenolic ingredient eugenol (commercially available). The synthesized bioconjugates were also bioactive, as reflected by their antibacterial effect due to their nano size which was confirmed by the various characterization techniques [12]. Encouraged by these results. Therefore, the present study deals with the anticancer effect of silver nano-bio conjugates synthesis from Piper betle leaf extract and its active phenolic component, eugenol.

Synthesis of silver nano-bio conjugates
Based upon the earlier research conducted in our laboratory, the method for nanobioconjugates synthesis was optimized and characterized, which confirmed that the sunlight exposure was rapid in synthesis [12]. The nanobioconjugates were synthesized by adding methanolic extract of Piper betle extract (PE) or diluted purified 100µg of eugenol (EU) to 1 mmol silver nitrate (Sigma) solution in the ratio of 10:90. The mixture of extract or compound and silver nitrate solution was exposed to bright sunlight (12500-13500 lux) for 20 min. After the exposure period, the mixture was centrifuged at 3500g for 20 min and washed thrice with deionized water. The nanobioconjugates were dried and dissolved in the DMSO for the further study.

Culturing of KB cells
The oral cancer cell line (KB) (Plate 1) were procured from National Center for Cell Sciences (NCCS), Pune, and used in this study. The cell lines were cultured and maintained in DMEM (Gibco) with 10% FBS (PAA) and 1X penicillin-streptomycin (MP Biomedicals) as per standard procedures and maintained in a CO2 incubator (Innova, UK). The confluent grown cells were trypsinized using Trypsin-EDTA (PAA) and suspended in a fresh complete medium. The flasks were incubated in a CO2 incubator (Innova, UK) with 95% humidity and 5% CO2. For the experiments, the cells were counted using hemocytometer and 105 live cells/per milliliter were seeded for the further experiments.

Culturing of buccal cells
The protocol of collection and the use of non-cancerous cells from healthy human volunteers was scrutinized and approved by the Institutional Human Ethics Committee (AUW/IHEC-14-15/XPD-08).
The buccal cells (Plate 1) were collected from healthy human volunteers, who were not on any medication in the age group of 20-30 y. The mouth was rinsed with 5 ml of Listerine; then sterile soft wood scrapers were used to scrape the buccal cells from the buccal cavity. About 15 ml of a sterile saline was used to rinse the mouth, which was collected in a sterile beaker, the scraper was also washed with 10 ml of sterile saline into the same sterile beaker. The cells were centrifuged at 300g for 5 min and the pellet was resuspended in 1 ml of sterile PBS. The suspended cells were treated with 1X penstrep (Penicillin-Streptomycin, from a 100X stock (MP Biomedicals, USA)), 12 µl/ml fluconazole (Cipla) and incubated for 30 min to remove any contamination. The cells were centrifuged at 300g for 5 min and washed twice with sterile PBS. Then the buccal cells were resuspended in complete DMEM (supplemented with 2% FBS, 1X Penstrep and 1.2µg/ml fluconazole) in the cell culture flask. The cells were incubated in a CO2 After 24 h of incubation, the buccal cells were transferred into a complete medium with 5% FBS. Again, the buccal cells were incubated for another 24 h and the cells were collected by trypsinization, centrifuged as earlier and the cell count was determined using a hemocytometer. The live healthy cells (105 cells/ml) were seeded in a 6-well plate with complete medium and with nano bio conjugates synthesized from betel leaf extract, their respective pure compound, and their respective non-nano raw material. The exposure was done at 37 ºC for 24 h. The cells were then harvested and centrifuged at 4000 rpm for 5 min, washed twice with sterile PBS and resuspended in PBS for the viability and staining analysis. chamber (Napco, UK) for 24 h.

Plate 1 Anticancer effect of nano bio conjugates
The anticancer activity was analyzed by cell viability and staining techniques. The cell viability was determined by the MTT [3(4,5dimethly-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide] reduction assay [13] and the apoptosis was evaluated by the AO/EtBr staining method [14] using a fluorescent microscope (Nikon, Japan) using B2A filter at 40X magnification. Live and apoptotic cells were counted and discriminated to calculate the apoptotic ratio. Live cells appeared uniformly green. Apoptotic cells incorporate the ethidium bromide and show the chromatin condensation and nuclear fragmentation. The apoptotic ratio was calculated using the following formula.

Apoptotic ratio = Number of apoptotic cells Number of normal cells
The assay and staining method were carried out in both the cancerous and non-cancerous cells. The following treatment groups were set up: • Untreated (negative) control cells • Cells treated with leaf extract/silver nanobioconjugates of P. betle • Cells treated with eugenol/silver nanobioconjugates of eugenol The parameters analyzed in this study were subjected to statistical analysis using SigmaStat (Version 3.1) statistical software. Statistical significance was determined by one-way ANOVA and the standard deviation value falls within±1 to±7.
Cell cycle analysis was carried out by flow cytometry, to determine the phase of cell cycle arrest [15]. The cells were analyzed for the populations of sub-G0, G0/G1, S and G2

RESULTS
/M phases of the cell cycle by the software of flow cytometry (BD FACSVerse, USA).
The MTT assay was used to determine the influence of silver nanobioconjugates on the viability of oral cancer cells along with the non-transformed buccal cells. A dose and time dependent effect of silver nanobioconjugates synthesized from Piper betle leaf extract (PAgNP) and eugenol (EAgNP) was studied. A wide range of both silver nanobioconjugates (2.5µg to 50µg/ml) and their extract alone. Based on the literature reports 50µM/ml of the active component, eugenol [16] was used in the study, at the exposure period of 24 h. The Piper betle leaf extract, eugenol, and their nanobioconjugates showed a dose-dependent decrease in the viability of KB cells. The results showed that the viability was lower in the treatment of silver nanobioconjugates groups when compared with the extract and compound alone ( fig. 1(i), 1(ii)). These observations show that the anticancer effect of both Piper betle leaf extract and its active phenolic component, eugenol, can be increased several folds by administering them as silver nanobioconjugates. The extent of cell death induced by the nanobioconjugates was very high at 24 h exposure period, therefore, the shorter exposure period of 16 h was studied. Similarly, as 24-h exposure results were obtained, KB cells showed high toxicity against all the dose levels ( fig. 2). Again the silver nanobioconjugates with the dose range of 2.5µg and 5µg/ml were tested for 12 h period. The results showed about 50% toxicity level, therefore, the 2.5µg concentration of silver nano bio conjugates was taken for the further study.

Fig. 2: Effect of silver nano bio conjugates synthesized from Piper betle leaves and their active component eugenol on the viability of KB cells (16 and 12 h treatment) The values are mean±Standard deviation of triplicates
From the wide range of doses 2.5µg, 20µg and 50µg/ml of silver nano bio conjugates synthesized from leaf extract and eugenol were selected for testing the cytotoxicity in the primary cultured buccal cells. The results ( fig. 3(i), 3(ii)), revealed that both the nano bio conjugates did not affect the viability of the buccal cells at the various dose level tested, which confirmed the differential effect of Piper betle leaf extract and its phenolic component on cancerous and non-cancerous cells of the oral cavity.

Fig. 3: Effect of silver nano bio conjugates on human buccal cells the values are mean±Standard deviation of triplicates
Following with the results of MTT assay, the type of cell death occurs in the cancer cells was studied by the AO/EtBr staining using a fluorescent microscope. From the microscopic study, it could be observed that the type of cell death observed was predominantly apoptosis. The number of apoptotic cells per 100 cells was counted in each treatment group, and the apoptotic index was calculated. From the results, it was observed that the leaf extract and its active component eugenol mediate the apoptosis process, whereas in the nanoform the apoptotic ratio was increased significantly (table 1 and Plate 2) The arrow marks in the plate 2 indicating the apoptotic cells.

10±2
The values are mean±Standard deviation of triplicates, a Statistically significant (P<0.05) compared to untreated control, b Statistically significant (P<0.05) compared to corresponding unconjugated extract/compound, c Statistically significant (P<0.05) compared to the corresponding extract/compound/group

Plate 2: Effect of extract / Compound / silver nano bio conjugates on the KB cancer cells and non-cancerous buccal cells, PE -Piper betle leaf extract, EU -Eugenol, PAgNP -Silver nano bio conjugates from betel leaves, EAgNP -Silver nano bio conjugates from eugenol
The events of the cell cycle in the exposed cancer cells was analyzed by flow cytometry. The results showed that the nanobioconjugates exhibited the anticancer effect by shifting the cells into S phase, and G2/M phase compares with the non-conjugate betel extract and eugenol ( fig. 4). The results obtained supported the earlier results obtained by MTT assay and staining technique, which proved that the effect of betel leaf extract and eugenol was more when they were administered as silver nano bio conjugates in the oral cancer cells.

DISCUSSION
The past ten years of research using metal nanoparticles such as silver, gold, copper, iron, titanium and ruthenium in preclinical research has shown promising anticancer properties [17]. Among the metal nanoparticles, silver and gold have been an attractive frontier of nanoparticle research because of their unique physicochemical properties in biomedical applications, such as bioimaging, biosensing, antimicrobial agents and cancer therapy [18].
Our results showed the strong anticancer activity of the silver nano bio conjugates synthesized. The extent of the anticancer effect was significantly more in the nano bio conjugates than their nonconjugated forms, proving that the anticancer activity of the test materials used can be enhanced markedly by preparing silver nano bio conjugates. The in vitro cytotoxicity assays have long been used for obtaining data on cell viability. Among these, MTT cell survival assay is used for analyzing and validating the cytotoxic potential of compounds [19]. Various reports on the anticancer activity of silver nanoparticles of plant-mediated extracts are available in the literature. The AgNPs synthesized from the ethanolic extract of rose petals showed a dosedependent decrease in the viability of human colon cancer HCT 15 cells [20]. Vasanth et al. [21] showed that the AgNPs of stem bark extract of Moringa oleifera possessed potent anticancer activity in human cervical carcinoma cells.
Followed by the cell viability assay, AO/EtBr staining was used to confirm the type of cell death caused by the silver nano bioconjugates, which was observed to be apoptosis in oral (KB). AO is a fluorescent dye that stains both dead and live cells, whereas EtBr stains only the dead cells, due to lost membrane integrity. Morphological changes have been reported in MCF-7 breast cancer cells treated with silver nanoparticles from Achillea biebersteinii using AO/EtBr staining [22]. Another study illustrated that nanosilver showed a stronger inhibitory effect on the liver cancer cells (HepG2) compared to the primary liver cells of mice, which was also observed using AO/EtBr staining [23]. In another study, silver nanoparticles of polyvinylpyrrolidone did not show any cytotoxicity in bone marrow cells, erythrocytes or human keratinocytes by AO/EtBr staining [24]. These reports lend further credibility to the results observed in the present study.
The silver nano bio conjugates showed a higher shift of cells from G0/G1 phase to the S phase and G2/M phase compared to the extracts/compounds in KB cells. These results clearly showed the induction of apoptosis by the silver nano bio conjugates, which was higher than the respective non-conjugated forms. The arrest of cells in G2/M phase has been recognized as representing a high level of cytotoxicity in several literature reports. The silver nanoparticles synthesized from ethanolic extracts of Phytolacca decandra, Gelsemium sempervirens, Hydrastis canadensis and Thuja occidentalis showed the highest peak in G2/M phase, which was suggested to indicate cytotoxic effect in human amelanotic melanoma (A375 cells) [25]. Similarly, the silver nanoparticles synthesized from mint, ginger, and coffee in HepG2 (hepatocellular liver carcinoma) and HeLa (human cervical cancer) caused cell cycle arrest in the G2/M phase [26]. Our results are in agreement with these reports. Thus, the silver nano bio conjugates synthesized from the plant source selected in this study exhibit stronger anticancer activity than the extracts and compound in their unconjugated form.

CONCLUSION
The findings of the present study, thus, validate and strengthen the method of synthesis of silver nano-bio conjugates from Piper betle leaves and their major polyphenol (eugenol) using a rapid, inexpensive and eco-friendly method. The anticancer properties of silver nano-bio conjugates were higher than their respective unconjugated forms, showing that the efficacy of their anticancer principles can be increased markedly by conjugating with silver to form nano bio conjugates.

CONFLICTS OF INTERESTS
Declared none


Genotoxic Effect of Chlorpyrifos and Cypermethrin in Albino Rats

This study was designed to evaluate genotoxic effect of chlorpyrifos and cypermethrin, singly and in combination, in albino rats. Thirty-six albino rats were randomly divided into three groups (A, B and C) of four rats per treatment (chlorpyrifos, cypermethrin and chlorpyrifos+ cypermethrin). Group A was the control and received distilled water and feed only. Groups B and C received 5 and 10 mg kgG b.wt. of each treatment, respectively. Treatment was administered via oral gavage and lasted for a period of 65 days. The 24 h after administering the last dosage, rats were sacrificed and bone marrow samples were collected for evaluation. The result revealed that pesticide treatment reduced polychromatic erythrocytes and the ratio of polychromatic erythrocytes to normochromatic erythrocytes significantly, while normochromatic erythrocytes and micronucleated polychromatic erythrocytes increased significantly (p<0.05) compared with the control. However, the treatment had no significant (p>0.05) effect on micro-nucleated normochromatic erythrocytes. These findings therefore indicate that chlorpyrifos and cypermethrin, singly and especially in combination, could be mutagenic.

INTRODUCTION
The use of pesticides has substantially improved the social and economic well being of inhabitants of developing countries through significant increase in food production and the effective mitigation of vector borne diseases. At the same time, however, public concerns over the indiscriminate application of pesticides and their possible adverse effect on animal and human health, as well as the environment has risen remarkably. Worldwide application of these chemicals is increasing continually as they have become a major part of the ecosystem. About 5.6 billion pounds of active pesticide ingredients are used yearly throughout the world (USEPA., 2001). Post market surveillance has shown that about 25 million agricultural workers globally experience unintentional pesticide poisoning annually (Alavanja et al., 2004).
It has been reported that pesticides available in the market may cause cancer in humans (Bonner et al., 2005). Zahm and Ward (1998), showed that cancers such as leukemia, neuroblastoma, sarcoma, lymphoma and cancer of the brain and testes are linked to pesticide exposure. These chemicals may also alter the genetic materials of organisms which may in turn affect them and their off spring (Manjula et al., 2006). Pesticides are reactive compounds that tend to form covalent bonds with various nucleophilic centres of cellular biomolecules, including DNA (Simoniello et al., 2008;Poletta et al., 2009). Because of their biological activity, the indiscriminate Res. J. Mut., 6 (1): [31][32][33][34][35]2016 se of these chemicals may cause undesired effects in humans. For instance, the induction of DNA damage can potentially lead to adverse reproductive conditions, cancer and many other chronic diseases (Meinert et al., 2000).
Since, mutation occurs spontaneously or under the influence of certain external factors like broad spectrum chemicals including pesticides, genotoxicity testing becomes necessary to assess the mutagenicity of chemicals in order to protect the human gene pool and identify potential carcinogens. To assess the genetic damage induced by physical and chemical agents including pesticides, various test systems have been described in bacteria, in mammalian cells in vitro and in plants (Celik et al., 2005). Arguably, the most reliable genotoxicity evaluation for human health risk is conducted in mammals by inducing chromosomal aberrations and micronucleus. In view of this premise, this present study was designed to assess the genotoxic effect of chlorpyrifos and cypermethrin, singly and in combination in bone marrow cells of male albino rats. Chlorpyrifos and cypermethrin were purchased from Department of Agrochemicals Ministry of Agriculture, Calabar, Nigeria.

MATERIALS AND METHODS
Experimental animals/procedure: Thirty six sexually mature male albino rats of body weight ranging from 170-200 g were purchased from the Department of Physiology, University of Calabar, Nigeria. They were kept in aluminum cages covered with wire mesh. They were fed with growers mash and allowed unrestricted access to clean water. The rats were allowed to acclimatize for one week. They were handled in accordance with the guidelines for care and use of laboratory animals as modified by the Department of Animal Genetics research committee.
The treatment was laid out in a 3×3 factorial experimental format using Completely Randomized Design (CRD). Group A serve as the control and received only distilled water. Groups B and C were administered 5 and 10 mg kgG 1 b.wt. of each treatment, respectively. Treatment was administered through oral gavage and lasted for a period of 65 days. Animals were sacrificed under chloroform anesthesia 24 h after the last dose was administered.
Bone marrow micronucleus assay: This method evaluates the genotoxicity of any mutagen. It has been accepted as a standard and a reliable in vivo assay to screen chemicals for their genotoxicity (Hodge and Sterner, 1956). The method described by Schmid (1976) was modified slightly and used to analyze micronuclei (MN) in polychromatic erythrocytes (PCEs) and normochromatic erythrocytes (NCEs) of rat bone marrow.
The animals were sacrificed, lower abdomen and limbs were incised and the femora were cleaned and separated from the hip joint. The ends of the femur were trimmed and a blunt needle was pushed to pierce the marrow cavity. Bone marrow was flushed into a tube containing 0.9% saline. Then, the suspension was made up to 5 mL and centrifuged at 1500 rpm for 10 min. The clear supernatant was discarded, while 2-3 drops of fetal calf serum were added to the pellet and mixed thoroughly. Smears were made on sterile coded slides using a drop of the suspension. The slides were air dried, fixed in absolute methanol and stained using May-Grunwald Giemsa method (D'Souza et al., 2005). Micronuclei were identified as dark blue staining bodies in the cytoplasm of polychromatic and normochromatic erythrocytes. Two thousand PCEs and NCEs each were scored per animal for the presence of MN using light microscope. The frequency of micro-nucleated cells was expressed in percentage (Marzouk et al., 2012).
Statistical analysis: All data obtained were subjected to analysis of variance (ANOVA) using PASW Version 18.0. Least Significant Difference (LSD) was used to separate the means that were significant at p<0.05.
The different pesticide treatment reduced PCE significantly. The least numerical value for PCE (38.75%) was observed in rats administered 10 mg kgG 1 b.wt. of chlorpyrifos+cypermethrin, compared with rats in the control group (50.25%) which was significantly higher. Meanwhile, pesticide treatment increased NCE significantly, though the mean value observed in rats of the control group was not significantly different from those recorded in treated rats. A similar trend was noticed wherein pesticide treatment increased MPCE in rats treated with pesticide. Rats in the control group had 1.5% as mean value of MPCE, whereas, the highest mean value (5.0%) was recorded in rats administered 10 mg kgG 1 b.wt. of chlorpyrifos+cypermethrin (Table 1).
However, pesticide treatment reduced P/N ratio significantly. Rats in the control group had the highest P/N ratio (1.12) compared with those treated with pesticide. It was also observed that rats administered 10 mg kgG 1 b.wt. of chlorpyrifos+cypermethrin had the lowest P/N ratio (0.66), although this was not significantly different from values in other groups except those in the control group (Table 1).

DISCUSSION
Application of pesticides is often regarded as an easy, cheap and quick means of eradicating pests. The use of these chemicals has scaled up production of crops as well as the control of vectors and diseases. Consequently, indiscriminate application of these chemicals has given rise to serious health and environmental concerns.
Pesticides have been implicated to alter the genetic materials of organisms they come in contact with, which could in turn affect them and their off spring (Manjula et al., 2006). Mutation occurs spontaneously or under the influence of certain physical or chemical agents such as pesticides, therefore, genotoxicity testing becomes necessary to assess their mutagenicity, identify potential carcinogens and protect the human gene pool. Micronuclei assay is an important tool used to  represent the consequence of chromosomal aberrations induced during the preceding mitotic divisions in erythroblasts (Matter and Grauwiler, 1975). Exposure to genotoxic substances damages chromosomes or components of the mitotic spindle leading to the formation of micronuclei. Thus, micronuclei may be formed from chromosomal materials fragmented during mitosis and not incorporated in the daughter nuclei at completion of telophase (Asare et al., 2012). In the present study, the result obtained revealed genotoxic effect of chlorpyrifos and cypermethrin in albino rats. Result for micronucleus assay showed that pesticide treatment had significant (p<0.05) effect on PCE, NCE, MPCE and P/N ratio but had no significant (p>0.05) effect on MNCE. It was further observed that pesticide treatment reduced PCE and P/N ratio significantly and increased NCE and MPCE compared with the control. The frequency of PCE in bone marrow cells provides an index of mitotic activity (Jayashree et al., 1994). Decline in PCE could be due to the suppression of proliferative activity of the bone marrow or suppression of erythrocyte production (Manjula et al., 2006). This reduction also suggests cell death. The formation of micronuclei demonstrates that a considerable amount of genetic information is no longer available to the cell and thus results in cell death (Jayashree et al., 1994). Increase in frequency of micro-nucleated cells reflects the damage caused by agents that induce chromosome breakage and non-disjunction. Since, these effects have been correlated with tumor initiation, the micronucleus test is appropriate for screening potentially genotoxic environmental agents (Jayashree et al., 1994). Meanwhile, reduction in P/N ratio due to pesticide treatment indicates that they have cytotoxic effect (Manjula et al., 2006). The result of this study is in agreement with the findings of Manjula et al. (2006) who reported that endosulfan increased MPCE and reduced PCE and P/N ratio significantly at 9 and 12 mg kgG 1 b.wt. Prasad et al. (2009) also reported that intraperitoneal administration of glyphosate at 25 and 50 mg kgG 1 b.wt., increased micronuclei induction significantly in rats. Furthermore, Marzouk et al. (2012) demonstrated that repeated administration of tribenuron-methyl increased the frequency of micronuclei formation in bone marrow of rats. Celik et al. (2005) also demonstrated that lambda-cyhalothrin induced a significant dose-related increase in micronuclei formation in bone marrow of Wistar rats at concentrations of 3.06 and 6.12 mg kgG 1 b.wt. Similarly, Kocaman and Topaktas (2009) reported that alpha-cypermethrin at 5 and 10 μg mLG 1 induced micronuclei formation in human peripheral blood lymphocytes, thus suggesting that the insecticide has genotoxic potential. On the contrary, Saleh et al. (2010) reported that thiocyclam did not cause any significant increase in MPCE of rat bone marrow cells. In the same vein, Alshehri (2014) reported that there was no significant increase in the frequency of micronuclei formation in bone marrow cells of rats exposed to methidathion.
The findings of this study therefore indicate that chlorpyrifos and cypermethrin, singly and in combination, have genotoxic potential in albino rats and thus could be mutagenic.