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Lead toxicity on non-specific immune mechanisms of freshwater fish Channa punctatus
Accepted Manuscript Title: Lead toxicity on non-specific immune mechanisms of freshwater fish Channa punctatus Author: Nilantika Paul Samujjwal Chakraborty Mahuya Sengupta PII: DOI: Reference:
S0166-445X(14)00095-2 http://dx.doi.org/doi:10.1016/j.aquatox.2014.03.017 AQTOX 3795
To appear in:
Aquatic Toxicology
Received date: Revised date: Accepted date:
26-6-2013 19-3-2014 20-3-2014
Please cite this article as: Paul, N., Chakraborty, S., Sengupta, M.,Lead toxicity on nonspecific immune mechanisms of freshwater fish Channa punctatus, Aquatic Toxicology (2014), http://dx.doi.org/10.1016/j.aquatox.2014.03.017 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Lead toxicity on non-specific immune mechanisms of freshwater fish Channa punctatus
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Nilantika Paul, Samujjwal Chakraborty and Mahuya Sengupta*,
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Department of Biotechnology, Assam University, Silchar-788 011, Assam, India
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*Corresponding author E-mail: [email protected]
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Tel.: +91 9435171903
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Fax: +91 3842 270806
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Abstract
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Lead has no known role in the body that is physiologically relevant, and its harmful effects are myriad. Lead from the atmosphere and soil ends up in water bodies thus affecting the aquatic
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organisms. This situation has thus prompted numerous investigations on the effects of this metal on the biological functions of aquatic organisms, particularly on immune mechanisms in fish. This paper addresses the immunotoxicologic effects of lead acetate in intestinal macrophages of freshwater fish Channa punctatus. Fish were exposed to lead acetate (9.43 mg/L) for 4 days. When checked for its effects on macrophages, it was noted that lead interfered with bacterial phagocytosis, intracellular killing capacity and cell adhesion as well as inhibited release of antimicrobial substances like nitric oxide (NO) and myeloperoxidase (MPO). On giving bacterial challenge with Staphylococcus aureus to intestinal macrophages of both control and lead treated groups, the macrophages showed significantly higher concentration of viable bacteria in the
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intracellular milieu in lead treated group as compared to control. We also report that in vivo exposure to lead acetate inhibits phagocytosis, which is evident from a reduced phagocytic index of treated group from that of the control. The amount of MPO and NO released by the control
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cells was also reduced significantly upon in vivo lead treatment. The property of antigenic adherence to the macrophage cell membrane, a vital process in phagocytosis, was significantly
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decreased in the treated group as compared to control. Severe damage in intestinal epithelium,
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disarrangement and fragmentation of mucosal foldings was observed in lead treated group when compared with the untreated group. The present results also showed decreased tumor necrosis
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factor- alpha (TNF-α) level upon metal exposure in sera as well as cell lysate of lead exposed fish thus, implicating both MAPK signalling pathways as well as NFκβ signalling. We thus
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conclude that lead affects the general immune status of C. punctatus and renders the fish
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immunocompromised and susceptible to pathogens.
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Keywords: lead, Channa punctatus, immunocompromised state, oxygen- dependent killing mechanisms, TNF-α, Scanning Electron Microscope.
Abbreviations:
HBSS, Hanks balanced salt solution; LPS, lipopolysaccharide; FBS, Fetal
Bovine Serum; DPBS, Dulbecco’s Phosphate Buffer Saline; SDS, Sodium Dodecyl Sulphate.
1. Introduction
Chronic contamination of aquatic environment by heavy metals is a severe problem since these pollutants invariably persist in the environment. Heavy metal contamination may have 2
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devastating effects on the ecological balance of the environment and a diversity of aquatic organisms (Velez and Montoro, 1998; Fang, 2004; Ashraj, 2005). The natural aquatic systems are extensively being contaminated with heavy metals released from domestic, industrial and
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other man-made activities (Conacher et al., 1993; Tafalla et al., 1999). These contaminants of aquatic environment not only endanger the survival and physiology of the organisms, but also
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induce genetic alterations which may lead to mutations and cancer (Russo et al., 2004).
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Lead (Pb) is an immunotoxicant which through exposure results in changes in immune function and has the potential to adversely affect human health. Many of its physical and chemical
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properties such as softness, malleability, ductility, poor conductivity and resistance to corrosion, have favored that man had been using lead and lead compounds since ancient times for a great
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variety of applications. The major hazard in industry arises from the inhalation of dust and fume
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of lead but the organic compounds may also be absorbed through the skin. It induces a broad
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range of physiological, biochemical and neurological dysfunctions in humans (Mobarak and Sharaf, 2010). Lead is widely used in several industrial processes such as clothe tinge, varnish,
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pesticide, explosives, batteries and painting manufacturing (Johnson, 1998) and could be responsible for death or sub lethal changes in reproduction, growth and behavior of the fishes (Burdena et al., 1998).
Like all vertebrates, fish possess a wide array of defense systems to protect themselves against heavy metals. Teleost fish have proved to be good models to evaluate the toxicity and effects of contaminants on animals, since their biochemical responses are similar to those of mammals and of other vertebrates (Sancho et al., 2000). C. punctatus is a freshwater teleost fish and was selected as an experimental model because of its wide availability round the year, and
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adaptability to laboratory. Intestine is the main route through which toxicants enter into the body and thus one of the first sites which comes into contact with the toxicants. Any damage to the intestinal lining can be a good indicator to the toxicity of the xenobiotic to biological system
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(Patel and Bahadur, 2011).
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Ferraro et al., (2004) and Ramsdorf et al., (2008) established that lead acetate induces micronucleus (MN) , chromosomal aberrations (CA) and causes DNA damage showing a
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significant increase of tailed nucleoids in the erythrocytes of fishes. Devi and Banerjee (2007)
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represented lead nitrate induced damage in the histology of air-breathing organs (ABO) of Channa striata in their study. Lead acetate induced micronucleus on cells of Carassus auratus
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auratus was also evaluated by Cavas (2008). Osman et al., (2009) established that lead acetate induces histopathological changes in gills, liver, kidney and spleen of Oreochromis niloticus.
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Studies of Mobarak and Sharaf (2010) revealed that lead acetate exposure resulted in various
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histopathological changes in the gills, liver, hepatopancreas, pancreas, stomach and intestine of
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Poecilia latipinna. Ahmed et al., (2011) reported that lead chloride induced DNA damage using alkaline single cell gel electrophoresis in liver tissue of Anabas testudineus. There are evidences of lead induced histopathological changes in liver, gills, intestine, kidneys and spleen of different fish models. In fish, the innate response has been considered an essential component in combating pathogens due to limitations of the adaptive immune system, their poikilothermic nature, their limited repertoire of antibodies and the slow proliferation, maturation and memory of their lymphocytes (Whyte, 2007). IL-1β and TNF-α have been detected in teleost fish species and is involved in the regulation of immunity (Subramaniam et al., 2002) causing the activation of macrophages, leading to increased respiratory activity, phagocytosis and nitric oxide production (Tafalla et al., 2001). High density fish farming and chemical contaminants in water, 4
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especially chronic exposure to toxic pollutants can lead to decreased immunity. Assays of macrophage function have been used as indicators of fish health (Warinner et al., 1988). Studies had showed that contaminants like lead can affect the phagocytic activity of macrophages in
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Oreochromis niloticus (Omima, 2010).
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However the influence of lead acetate on the non- specific immune system of C. punctatus is still unclear. Thus a better understanding of the mechanisms through which heavy metals influence
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the non-specific immune system is necessary to appreciate the many complex interactions
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between environmental contaminants and the system’s susceptibility to infectious diseases. The aim of the present study was to evaluate the immunotoxicologic effects of lead acetate in
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intestinal macrophages of C. punctatus.
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2.1. Biological material
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2. Materials and methods
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Experiments were performed on Channa punctatus of sizes varying between length 12.08±0.41 and 14.5±0.52 cm and weight 18.07±0.75 and 22.5±0.25 g. Animals were purchased from a local market; upon arrival at the laboratory, the animals were housed in glass aquarium and kept for at least 5-6 days to allow for acclimatization. Only healthy fish that were not diseased, as determined by general appearance (color, skin luster, eyes and behaviour), were used for the studies.
2.2. Exposure After acclimatization, two groups of fish, one for control and the other for lead-treated, were established in a gently aerated semi-static system. Each group consisted of three tanks which 5
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served as triplicates. Each tank housed five fish (n=5). One group was exposed to 9.43 mg/L (2.9 M), 1.02% of 96h LD50 value [925 mg/L] (Devi and Banerjee, 2007) lead acetate solution (prepared in autoclaved distilled water) for 4 days. The other group served as control which was
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kept unexposed to lead acetate. The tank water was changed on a regular basis, the temperature being in the range between 22 and 28ºC, pH 7.2, dissolved O2 6.5 mg/L (90% saturation) and
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salinity ranging from 0.09 to 0.17 mg/l. Fish were fed with minced goat liver and water was
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renewed every 24h. Feeding was allowed to both experimental and control fish throughout the tenure of experiments. All assays were performed in triplicates. The fish were housed, treated
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2.3. Isolation of intestinal macrophages
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and sacrificed following the guidelines of the Institutional Ethics Committee, Assam University.
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The fish were dissected and the whole gut of the fish were isolated, immediately placed in
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Leibovitz medium (L-15) supplemented with heparin (10 IU/ml) and fetal bovine serum (FBS) (2%), and then homogenized in ice cold condition. Cell suspension was then transferred to tubes
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and kept in ice for the settlement of cell debris. The supernatant was layered over Ficoll (45%) and subjected to density- gradient centrifugation (Chung and Secombes, 1998). The band of macrophage-enriched fraction at the interface is collected, washed and resuspended in L-15 medium containing FBS and allowed to adhere on plastic surface. The adherent cells of both exposed and unexposed group were collected and tested for viability at different time intervals as determined by trypan blue dye exclusion technique. Tissue samples showing cell viability higher than 95% were further processed for various analyses.
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2.4. Phagocytosis assay: A volume of 100 µl of cells from both control and exposed groups were allowed to adhere
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separately on glass slides for one hour whereas non-adherent cells were washed out with Dulbecco’s phosphate buffered saline (DPBS) (1X). To the glass slides containing the adhered
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macrophages 10% heat killed Staphylococcus aureus was added and incubated for 3 h at 37ºC which were then washed with DPBS (1X) and dried. The cells were at last fixed in 50%
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methanol, stained with Giemsa, observed under oil immersion microscope and counted for
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number of bacterial cells ingested (Czuprynski et al., 1984). Phagocytosis is expressed as Phagocytic index (%) = (percentage of macrophages containing bacteria) × mean number of
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bacteria per macrophage).
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2.5. Intracellular killing assay:
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Bacteria were incubated with macrophages in L-15 FBS for 20 min at 37oC. After various time
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intervals, samples were plated onto nutrient agar to determine the number of viable intracellular bacteria (Leigh et al., 1986). Intracellular killing was expressed as the percentage decrease in the initial number of viable intracellular bacteria.
2.6. In vitro cell adhesion assay:
Cells were seeded separately for treated and control group in 96 well microtitre plates and allowed to adhere for different times. In time, wells were washed with Hank’s balanced salt solution (HBSS) and then 100 µl of 0.5% crystal violet in 12% neutral formaldehyde, and 10% ethanol was added to each well and incubated for 4 h to fix and stain the cells. Wells were washed and air dried for 30 min. Crystal violet was extracted from the macrophage adhered in 7
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the wells by lysing with 0.1% sodium dodecyl sulphate (SDS) in HBSS. Absorbance was measured spectrophotometrically at 570 nm. Cell adhesion was expressed as increased
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absorbance at 570 nm (Lin et al., 1997).
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2.7. Nitric oxide (NO) release assay:
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Cells (106 cells/ml) were suspended in DPBS and were stimulated with lipopolysaccharide or LPS (100 ng/ml).The cell free supernatants were used for nitric oxide release assay using Griess
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reagent. Readings were taken in a UV spectrophotometer at 550nm. The absorbance was plotted
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on a nitrate standard curve and NO release was expressed as µM (Saggers and Gould, 1989).
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2.8. Myeloperoxidase (MPO) release assay:
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A volume of 100 µl of cells from control and treated group were taken into microcentrifuge
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tubes and stimulated with LPS (100 ng/ml) for 1 hr at 37ºC and centrifuged at 13000 rpm for 10 min. The supernatants thus obtained from both the sets was recovered separately and kept at 20ºC until further use. The cell free supernatant was used for assay of the partial MPO release for both groups. The pellet that recovered from the two groups were lysed in 0.01% SDS and then centrifuged again. The supernatant was recovered as before for total MPO release assay. Subsequently 100 µl of cell free supernatant as well as from lysis of cells were reacted with 100 µl of substrate buffer and kept at 37ºC for 20 min, then the reaction was stopped by adding 100 µl of 2(N) H2SO4 and absorbance was measured at 492 nm (Bos et al., 1990).
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2.9. Ultrastructural analysis of tissue by Scanning Electron Microscope The intestines from treated and untreated groups were excised and rinsed in heparinized saline,
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rinsed in 0.1 M cacodylate buffer at pH 7.5, infiltrated with 2.5% glutaraldehyde for 24 hr fixation at 4ºC, rinsed in buffer, trimmed into 8.0 mm squares and subjected to post-fixation in
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1% OsO4 in 0.1 M cacodylate buffer at pH 7.5 for 2 h and dehydrated through graded acetone. (Spurr, 1969). The mucosal surface of each tissue was mounted on metal stubs, coated with gold
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using a JFC-1100 (Jeol) ion sputter. Finally, the tissues were scanned with a JSM-6360 (Jeol)
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Scanning Electron Microscope at the Sophisticated Analytical Instrument Facility (SAIF),
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NEHU, Shillong, Meghalaya.
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2.10. Preparation of samples for cytokine analysis
b) From serum
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a) From cell supernatant (in vitro)
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a) Isolated cells were treated with LPS (5 ng/ml) with or without metal treatment. The cells were lysed and any particulate material was removed by centrifugation, supernatant collected and assayed immediately.
b) Peripheral blood was obtained from treated and untreated fish by heart puncture into 1.5 ml microcentrifuge tubes. After cooling overnight at 4ºC, serum was collected after centrifugation and used for cytokine analysis. 2.11. Cytokine analysis: The 96 well microtitre plates (Iwaki Glass, Japan) were coated with 100 µl of fish serum or cell supernatant diluted 10 times in phosphate buffer saline (PBS) and incubated in a moist chamber 9
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overnight at 4ºC. TNF- α rabbit polyclonal antibody was used as the primary antibody (Sigma, USA). Vectastain ABC- PO kit (Vector Lab., USA) was applied for ELISA and peroxidase ABTS substrate kit (Vector Lab., USA) was used for coloration. The plate was read at 490 nm on
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a microplate reader (BioRad). For the blank, physiological saline was used instead of serum.
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Fish sera from untreated groups served as control. All assays were performed in triplicate.
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2.12. Statistical analysis
The data were expressed as mean±S.E.M. Data were analyzed using Student’s t-test (two-sample
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assuming unequal variances) for determining the significant change over control values. The
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significance level was set at P<0.05.
3. Results
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Staphylococcus aureus
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3.1. Effect of lead acetate on phagocytic capacity of fish intestinal macrophages against
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Phagocytic index of the control group was found to be 21636.66±5.96 while that of lead treated group was found to be 10821.62±4.14 (P<0.01). [Fig:1] 3.2. Effect of lead acetate on intracellular killing capacity of fish intestinal macrophages On giving bacterial challenge (with S. aureus) to intestinal macrophages of both control and lead treated groups, bacterial load showed significantly higher concentration of viable bacteria at an interval of 15 min (P<0.004), 30 min (P<0.005), 45 min (P<0.002) and 50 min (P<0.007) in the heavy metal treated group as compared to control (Fig:2). 3.3. Effect of lead acetate on cell adhesion property of fish intestinal macrophage. 10
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Cell adherence was expressed as the decreased absorbance at 570 nm. After 30 min incubation, absorbance in lead-treated group was found to be 0.07±0.002 from 0.15±0.002 of control group (P<0.04). After 60 min incubation, absorbance in lead-treated group was found to be 0.13±0.002
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from 0.21±0.002 of control group (P<0.02) (Fig:3).
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3.4. Effect of lead acetate on nitric oxide release in fish intestinal macrophages
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NO released from lead-treated+ LPS stimulated intestinal macrophages was found to be
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(4.61±0.197) µM whereas that in control group was (50.94±0.94) µM (P<0.00005) (Fig:4). 3.5. Effect of lead acetate on myeloperoxidase release in fish intestinal macrophages
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Myeloperoxidase (MPO) release was studied and was found to decrease from (43.51±1.2) % in
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control to (24.42±1.6) % (P<0.02) in lead-treated group (Fig:5).
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3.6. Ultramicroscopic analysis of intestinal tissue by Scanning Electron Microscope
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The present results showed severe damage in epithelium, disarrangement and fragmentation of mucosal foldings and secretion of mucus [Fig: 6(c) and 6(d)] upon lead exposure. Untreated group showed normal epithelium with prominent mucosal foldings [Fig: 6(a) and 6(b)]. 3.8. Effect of lead acetate on TNF- α release from fish intestinal macrophage lysate TNF-α release in intestinal macrophages was studied and was found to decrease from 157 ± 1.28 pg/ml in control to 101 ± 1.14 pg/ml (P<0.02) in lead treated group respectively (Fig:7). 3.7. Effect of lead acetate on serum TNF- α levels
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TNF-α release was studied and was found to decrease from 218 ± 1.25 pg/ml in control to 155 ± 1.34 pg/ml (P<0.02) in lead treated group (Fig:8).
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4. Discussion Chronic lead exposure can affect proliferation of white blood lymphocytes. Macrophages play a
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critical role in body’s defense system by eliminating microorganisms from infected tissues
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(Gonsebatt et al., 1992). Phagocytosis by fish cells has been evaluated, both in vivo and in vitro, using different test particles (Estaban et al., 1998; Suzuki, 1986), including yeast cells (Bayne,
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1986; Hyder, 1983; Saggers and Gould, 1989). Macrophages recognize and engulf bacteria into phagosomes, which subsequently acidify. These phagosomes mature into phagolysosomes upon
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vesicle-mediated delivery of various antimicrobial effectors (Garin et al., 2001). From the
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phagocytic index of the present findings it is observed that intestinal macrophages from the
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control group phagocytose S. aureus at a higher rate, which is reduced upon lead exposure. This suggests that heavy metal exposed macrophages fail to perform their normal functions, thereby
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allowing foreign particles or pathogens to gain access to the body which is clearly depicted in the results of the present study.
Professional phagocytes have evolved and are known to internalize and kill as many antigens as possible. Normally, bacteria within macrophages are rapidly killed and degraded in the phagolysosome with the secretion of enzymes and toxic peroxides, making it difficult to dissect the mechanism of death (Slauch, 2011). The present study clearly illustrates a greater percentage of viable bacteria within the lead treated macrophages as compared to that of the control group
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which suggests that lead limits its intracellular killing activity after internalization of the pathogen.
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Chemotaxis is the process by which phagocytic cells are attracted by various molecules like leucocyte specific cell adhesion molecules (CAMs) and migrate to the sites of inflammation,
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causing tissue damage or immune reactions. Adherence of antigen to the macrophage cell
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membrane is a vital step in phagocytosis. Complex antigens, such as whole bacterial cells or viral particles, tend to adhere well and are readily phagocytosed (May and Machesky, 2001;
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Aderem, 2002; Underhill and Ozinsky, 2002). However, this property of cell adherence is significantly inhibited upon lead exposure as it is evident from a fall in absorbance at 570 nm
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with respect to control. Decrease in in vitro cell adhesion in lead intoxicated group could be
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primarily due to decreased expression of CAMs on the surface of macrophage cells.
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In recent years, NO has been shown to be a very important molecule in regulating immune functions as well as having a direct antimicrobial effect (Gunasegaran et al., 1993; Liew et al.,
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1990). It exhibits a wide range of important functions in vivo, acting as a releasing factor mediating vasodilation, as a neuronal messenger molecule, and as a major regulatory molecule and principal cytotoxic mediator of the immune system (Beckman and Koppenol, 1996; Dimmeler and Zeiher, 1997; Langston et al., 2001). Reactive nitrogen species (RNS), a family of antimicrobial molecules derived from nitric oxide (NO), are produced via the enzymatic activity of inducible nitric oxide synthase 2 (NOS2) (Fang, 2004; Nathan and Shiloh, 2000). The present results illustrate that the amount of NO production by the lead exposed group is strikingly less as compared to the control group. This finding may probably signify suppression of NOS2 expression and subsequent decrease in antimicrobial activity of macrophage.
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Microbicidals like MPO produced by macrophages are known to play an important role in cellular defenses against various bacterial infections. MPO can generate oxidants from H2O2 and a range of co-substrates, most notably chlorine (Foote et al., 1983) and nitrite (Eiserich, 1998).
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HOCl that is produced by MPO is strongly bactericidal (Klebanoff, 1967) and markedly increases the antibacterial potency of ROS (Rosen and Klebanoff, 1979; Rosen et al., 1990). This
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might indicate that MPO is important in host defense in fish. Insufficient release of MPO from
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lead treated macrophages may indicate that its bactericidal potency has been suppressed resulting
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in poor host defense mechanism.
Heavy metals not only deteriorate the physico-chemical equilibrium of the aquatic body, but also
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disrupt the food web and, bring about morphological, physiological and cytogenetic changes in
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the aquatic inhabitants (Yadav and Trivedi, 2009). The functions of teleostean digestive tract
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include digestion, nutrients absorption, hormone secretion, immune protection and water and salt transfers for hydro mineral homeostasis. It regulates energy and material exchange between the
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environment and the internal medium (Khojasteh, 2012). Thus knowledge of the intestinal ultrastructure and morphology is essential for understanding the related functional mechanisms. This study aims to investigate whether heavy metals like lead could induce morphological alterations in fish gut or not. Scanning Electron Microscopy (SEM) is an important tool that reveals information on the surface ultrastucture and morphological changes that may occur in biological tissues. Lead induced severe damages in intestinal epithelium, disarrangement and fragmentation of mucosal foldings, as observed in the present study, cause impairment of tissue organs which in turn has deleterious effects on its immune defense system. These results significantly correlate with the altered non-specific immune functions of the macrophages.
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Furthermore, lead induced suppression of TNF-α activity, as observed in the present study, may also contribute to the above results.
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Macrophages play an essential role for the initiation and activation of the innate immune system, by recognizing and releasing a large number of cytokines such as tumor necrosis factor- a (TNF-
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α), interleukin-1 (IL-1) and IL-6 (Nathan, 1987). The existence of TNF-α like protein in fish has
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been already reported by several authors. Zelikoff et al. (1990) reported the presence of TNF- α in macrophages of rainbow trout, Onchorhynchus mykiss. Hardie et al. (1994) have found that
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rainbow trout lymphocytes and macrophages can functionally respond to human recombinant TNF-α, which suggests that fish leucocytes may possess a specific TNF-α receptor that might
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have been highly conserved in evolution. In addition, TNF-α was one of the cytokines that was
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detected by Ahne, (1993) in the serum of virus infected carp. To determine whether heavy metals
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could alter the secretion of proinflammatory cytokines from macrophages, the release of TNF-α was quantified in lead treated fish serum as compared to control. Our results showed decreased
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TNF-α levels upon metal exposure in serum of lead exposed fish. TNF-α is a proinflammatory cytokine that affects tissue vasculature in inflammation and also induces the acute phase proteins from the liver. In fish, phosphorylcholine reactive protein (PRP), an acute phase protein homologous to C-reactive proteins, is expressed by the action of TNF-α (Szalai et al., 1994).
Serum levels of TNF-α was found to be significantly less in lead treated group than that of control group. This motivated us to investigate the cytokine release from the macrophages of the treated group stimulated with lipopolysaccharides (LPS) under in vitro conditions. Upon LPS
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stimulation, TNF-α levels in the macrophage supernatant was found to be significantly less in lead treated group than that of control group.
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Lipopolysaccharides (LPS) upregulate proinflammatory cytokines in macrophages, partly through a NF-κB-dependent process. The upregulation of secreted inflammatory cytokines by
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macrophages occurs upon the selective recognition and interaction of the pathogen-associated
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molecular patterns (PAMPs) that are conserved in bacterial species (e.g., lipopeptides, lipopolysaccharides, or flagellin) by the pattern recognition receptors (PRRs) such as the Toll-
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like receptor (TLR) proteins. Perhaps the best characterized example of this microbial recognition by TLRs is the recognition of lipopolysaccharides (LPS) from Gram negative
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bacteria by TLR4. The interaction between LPS and TLR4
signals through Interleukin-1
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Receptor-Associated Kinases (IRAKs), which subsequently signal through Mitogen Activated
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Protein Kinase Kinase 6 (MAPKK6) and the Inhibitor of Nuclear Factor-κB Kinase (IKK). MAPKK6 and IKK activation culminate in the activation of Activator Protein-1 (AP-1) and
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Nuclear Factor-κB (NF-κB) transcription factors, respectively, resulting in the transcriptional upregulation of several proinflammatory cytokines like TNF-α (Chang et al., 2012).
Our findings help in establishing the fact that lead acetate render the fish to an immunocompromised and anti-inflammatory state. Cytokine expression thus becomes downregulated, probably by the action of the heavy metals at multiple levels viz., PAMP recognition, receptor dysfunction and/or by affecting the cell-signalling network.
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Thus, in the present study, we have examined some of the cellular activities of intestinal macrophages isolated from lead acetate exposed fish, like cell adhesion, phagocytosis and intracellular killing of bacteria. These have been found to be directly related to the cellular
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morphology of the macrophages. Prolonged exposure to heavy metals alters the morphology and diminishes the functional capacities of intestinal macrophages, which can also be correlated, to
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their lowered killing capacity in lead exposed fish. In immune cells, which are rapidly
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proliferating and differentiating in response to bacterial challenge, both immune mechanism and cellular activation are critically important. Inhibition of these functions may result in similar
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degree of toxicity. The results in this study provide a baseline for subsequent studies on the role of inflammatory cells in the pathogenesis of bacterial insults during heavy metal intoxication.
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Since cell adherence was significantly decreased in that of lead exposed cells, it can be suggested
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that in vivo heavy metal intoxication may somehow change the shape, alter membrane signals
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and functionally inactivate the cells so that they migrate at a slower rate. Macrophages contain on their surfaces and also secrete proteolytic enzymes, active at the tissue pH that may be
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important in their ability to migrate in vivo. Numerous substances generated during inflammation, like cytokines and chemokines, have the capacity to enhance the speed of macrophage and orient the movement in the direction of an increased concentration gradient of the agent. Lead exposure may lead to alteration in the expression of cell adhesion molecules on the macrophage membrane as well as the proinflammatory cytokines within the macrophage milieu which further alters the shape and orientation of macrophages so that they migrate slowly and adhere to and engulf bacteria poorly. Reduced adherence of such phagocytic cells to vascular and capillary endothelial cell lining may go without any effective phagocytosis and intracellular killing of the pathogens.
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Similarly, for initiation of phagocytosis, contact between particulate antigen and phagocytic cell is important. Morphologic alteration of the (lead exposed) macrophages exposed to lead to both reduced cell adhesion as well as phagocytosis. Diminished rate of bacterial clearance from lead
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exposed fish reveals that bacteria may intracellularly survive for a prolonged period indicating susceptibility to severe opportunistic infectious diseases. Rate of bacterial clearance by tissue
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fixed macrophages recovered (isolated) from lead exposed fish may be related to pathogenesis of
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bacteria. The intracellular milieu provides slow growing organisms with an unoccupied niche where microbial competition is relaxed or non-existent. Additionally, an intracellular lifestyle is
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thought to shelter organisms from immune surveillance. However, there is little evidence to support this idea regarding S. aureus infection during lead intoxication. A variation in
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morphology and functional activities of macrophages including enzyme release suggests that the
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immune defense systems of C. punctata intestine are compromised by lead exposure at low
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concentrations, probably by compromising the molecular cross-talk between signaling molecules. Suppression of proinflammatory cytokine release (TNF and IL-1b) implicates the
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involvement of MAPK cascades and downregulation of NFkB genes.
A number of antimicrobial and cytotoxic substances produced by activated macrophages can destroy phagocytized microorganisms. When macrophages are activated with bacterial cell wall lipopolysaccharide (LPS), they begin to express high levels of myeloperoxidase and nitric oxide synthase enzymes as well as the proinflammatory cytokines. NO itself has antimicrobial activity and it can also combine with superoxide anion and peroxide radicals to yield other potent antimicrobial substances. In the present study, it was found that both MPO and NO release significantly decreases in the lead challenged groups as compared to that of control as does the
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level of the cytokines TNF and IL-1b. So, it can be suggested that lead exposed groups in the face of reduced MPO and NO secretion, are more prone to infections and cannot phagocytose efficiently and as a result, cannot clear the invading microorganism which may lead to diseased
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state upon bacterial invasion. Suppression of proinflammatory cytokine release (TNF and IL-1b) implicates the involvement of MAPK cascades and downregulation of NFkB genes that trigger
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these morphological, ultrastructural and functional alterations of the intestinal macrophages.
Conclusively, lead has a deleterious effect on the immune functions of fish, namely on its gut-
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associated lymphoid tissue (GALT). Macrophages constitute the first line of defense against antigenic insults in an organism. A variation in functional activities of macrophages reveals that
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the non- specific immune defense system of C. punctata intestine is compromised by lead
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exposure at low concentrations. The exposure to sub-lethal concentration of lead acetate caused
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suppression of the parameters (phagocytic activity, intracellular killing, cell adhesion, nitric oxide release, myeloperoxidase release and cytokine release) measured in the intestinal
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macrophage of C. punctatus, probably by compromising the molecular cross-talk between signaling molecules. Contamination of water bodies by numerous chemicals and non-essential elements, such as heavy metals like lead, is an unfortunate byproduct of a complex, industrialized, high-tech society. These results also suggest that immunological profiles of fish under heavy metal stress may serve as important bio-indicators in the monitoring of aquatic ecosystems.
Conflict of Interest The authors do not have any conflict of interest in the present work. 19
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Hyder, S.L., Cayer, M.L., Pettey, C.L., 1983. Cell types in peripheral blood of the nurse shark: an approach to structure and function. Tissue Cell 15, 437-455. Johnson, F.M., 1998. The genetic effects of environmental lead. Mutat Res 410, 123-140. Khojasteh, S. M. B., 2012. The morphology of the post-gastric alimentary canal in teleost fishes: a brief review. Int. J. Aq. Sc. 3, 17-27. 22
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Mobarak, Y. M. S., Sharaf, M. M., 2010. Lead acetate-induced histopathological changes in the gills and digestive system of silver sailfin molly (Poecilia latipinna). Int. J. Zool. Res. 7, 1-18. Nathan, C., Shiloh, M.U., 2000. Reactive oxygen and nitrogen intermediates in the relationship between mammalian hosts and microbial pathogens. Proc Natl Acad Sci U S A 97, 8841-8848. Nathan, C.F., 1987. Secretory products of macrophages. J Clin Invest 79, 319-326. Omima, A. S. A. Aboud., 2010. Impact of pollution with lead, mercury and cadmium on the immune response of Oreochromis niloticus. New York Sc. J. 3(9).
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Osman, M. M., El- Fiky, S. A., Soheir, Y. M., Abeer, A. I., 2009. Impact of water pollution on histopathological and electrophoretic characters of Oreochromis niloticus fish. Res. J. Environ Toxicol. 3, 9-23.
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Rosen, H., Klebanoff, S.J., 1979. Bactericidal activity of a superoxide anion-generating system.
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Spurr, A.R., 1969. A low-viscosity epoxy resin embedding medium for electron microscopy. J Ultrastruct Res 26, 31-43.
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Suzuki, K., 1986. Morphological and phagocytic characteristics of peritoneal exudate cells in tilapia, Oreochromis niloticus (Trewavas), and carp, Cyprinus carpio L. Journal of Fish Biology
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(Ictalurus punctatus Rafinesque) phosphorylcholine-reactive protein (PRP) in response to inflammatory agents, low temperature-shock and infection by the fungus Saprolegnia sp. Fish &
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Whyte, S.K., 2007. The innate immune response of finfish: A review of current knowledge. Fish and Shellfish Immunology 23, 1127–1151. Yadav, K.K., Trivedi, S.P., 2009. Sublethal exposure of heavy metals induces micronuclei in
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pp 86.
Figure Legends:
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Fig:1. Phagocytic activity in intestinal macrophages of fish treated with lead. Values are
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expressed as mean±S.E.M., significant difference with the control group (P<0.01).
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Fig:2. Intracellular killing capacity of control and lead exposed fish intestinal macrophages.
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Values are expressed as mean±S.E.M., significant difference with the control group (P<0.004). Fig:3. Cell adhesion capacity of control and lead exposed fish intestinal macrophages. Values are expressed as mean±S.E.M., significant difference with the control group at 30 minutes (P<0.04) and 60 minutes (P<0.02).
Fig:4. NO released from control and lead exposed fish intestinal macrophages. Values are expressed as mean±S.E.M., significant difference with the control group (P<0.00005). Fig:5. MPO released from control and lead treated fish intestinal macrophages. Values are expressed as mean±S.E.M., significant difference with the control group (P<0.02).
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Fig: 6. Ultramicroscopic changes in (a), (b) control and (c), (d) lead treated fish intestinal tissue. Fig: 7. TNF-α released from control and lead treated fish serum. Values are expressed as mean ±
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S.E.M., significant difference with the control group (P<0.02). Fig: 8. TNF-α released from control and lead treated fish intestinal macrophage lysate. Values
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are expressed as mean ± S.E.M., significant difference with the control group (P<0.02).
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(AQTOX-D-13-00340R1) Lead toxicity on non-specific immune mechanisms of freshwater fish Channa punctatus
Highlights:
Study of lead toxicity in intestinal macrophages of freshwater fish Channa punctatus.
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Lead alters bacterial phagocytosis, intracellular killing and cell adhesion.
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It also inhibits release of antimicrobials like nitric oxide and myeloperoxidase.
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Decreased TNF-α upon lead exposure implicates both MAPK and NFκβ signalling.
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Thus, lead immunocompromises C. punctatus rendering it susceptible to pathogens.
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S0166-445X(14)00095-2 http://dx.doi.org/doi:10.1016/j.aquatox.2014.03.017 AQTOX 3795
To appear in:
Aquatic Toxicology
Received date: Revised date: Accepted date:
26-6-2013 19-3-2014 20-3-2014
Please cite this article as: Paul, N., Chakraborty, S., Sengupta, M.,Lead toxicity on nonspecific immune mechanisms of freshwater fish Channa punctatus, Aquatic Toxicology (2014), http://dx.doi.org/10.1016/j.aquatox.2014.03.017 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Lead toxicity on non-specific immune mechanisms of freshwater fish Channa punctatus
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Nilantika Paul, Samujjwal Chakraborty and Mahuya Sengupta*,
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Department of Biotechnology, Assam University, Silchar-788 011, Assam, India
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*Corresponding author E-mail: [email protected]
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Tel.: +91 9435171903
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Fax: +91 3842 270806
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Abstract
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Lead has no known role in the body that is physiologically relevant, and its harmful effects are myriad. Lead from the atmosphere and soil ends up in water bodies thus affecting the aquatic
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organisms. This situation has thus prompted numerous investigations on the effects of this metal on the biological functions of aquatic organisms, particularly on immune mechanisms in fish. This paper addresses the immunotoxicologic effects of lead acetate in intestinal macrophages of freshwater fish Channa punctatus. Fish were exposed to lead acetate (9.43 mg/L) for 4 days. When checked for its effects on macrophages, it was noted that lead interfered with bacterial phagocytosis, intracellular killing capacity and cell adhesion as well as inhibited release of antimicrobial substances like nitric oxide (NO) and myeloperoxidase (MPO). On giving bacterial challenge with Staphylococcus aureus to intestinal macrophages of both control and lead treated groups, the macrophages showed significantly higher concentration of viable bacteria in the
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intracellular milieu in lead treated group as compared to control. We also report that in vivo exposure to lead acetate inhibits phagocytosis, which is evident from a reduced phagocytic index of treated group from that of the control. The amount of MPO and NO released by the control
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cells was also reduced significantly upon in vivo lead treatment. The property of antigenic adherence to the macrophage cell membrane, a vital process in phagocytosis, was significantly
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decreased in the treated group as compared to control. Severe damage in intestinal epithelium,
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disarrangement and fragmentation of mucosal foldings was observed in lead treated group when compared with the untreated group. The present results also showed decreased tumor necrosis
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factor- alpha (TNF-α) level upon metal exposure in sera as well as cell lysate of lead exposed fish thus, implicating both MAPK signalling pathways as well as NFκβ signalling. We thus
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conclude that lead affects the general immune status of C. punctatus and renders the fish
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immunocompromised and susceptible to pathogens.
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Keywords: lead, Channa punctatus, immunocompromised state, oxygen- dependent killing mechanisms, TNF-α, Scanning Electron Microscope.
Abbreviations:
HBSS, Hanks balanced salt solution; LPS, lipopolysaccharide; FBS, Fetal
Bovine Serum; DPBS, Dulbecco’s Phosphate Buffer Saline; SDS, Sodium Dodecyl Sulphate.
1. Introduction
Chronic contamination of aquatic environment by heavy metals is a severe problem since these pollutants invariably persist in the environment. Heavy metal contamination may have 2
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devastating effects on the ecological balance of the environment and a diversity of aquatic organisms (Velez and Montoro, 1998; Fang, 2004; Ashraj, 2005). The natural aquatic systems are extensively being contaminated with heavy metals released from domestic, industrial and
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other man-made activities (Conacher et al., 1993; Tafalla et al., 1999). These contaminants of aquatic environment not only endanger the survival and physiology of the organisms, but also
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induce genetic alterations which may lead to mutations and cancer (Russo et al., 2004).
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Lead (Pb) is an immunotoxicant which through exposure results in changes in immune function and has the potential to adversely affect human health. Many of its physical and chemical
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properties such as softness, malleability, ductility, poor conductivity and resistance to corrosion, have favored that man had been using lead and lead compounds since ancient times for a great
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variety of applications. The major hazard in industry arises from the inhalation of dust and fume
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of lead but the organic compounds may also be absorbed through the skin. It induces a broad
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range of physiological, biochemical and neurological dysfunctions in humans (Mobarak and Sharaf, 2010). Lead is widely used in several industrial processes such as clothe tinge, varnish,
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pesticide, explosives, batteries and painting manufacturing (Johnson, 1998) and could be responsible for death or sub lethal changes in reproduction, growth and behavior of the fishes (Burdena et al., 1998).
Like all vertebrates, fish possess a wide array of defense systems to protect themselves against heavy metals. Teleost fish have proved to be good models to evaluate the toxicity and effects of contaminants on animals, since their biochemical responses are similar to those of mammals and of other vertebrates (Sancho et al., 2000). C. punctatus is a freshwater teleost fish and was selected as an experimental model because of its wide availability round the year, and
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adaptability to laboratory. Intestine is the main route through which toxicants enter into the body and thus one of the first sites which comes into contact with the toxicants. Any damage to the intestinal lining can be a good indicator to the toxicity of the xenobiotic to biological system
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(Patel and Bahadur, 2011).
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Ferraro et al., (2004) and Ramsdorf et al., (2008) established that lead acetate induces micronucleus (MN) , chromosomal aberrations (CA) and causes DNA damage showing a
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significant increase of tailed nucleoids in the erythrocytes of fishes. Devi and Banerjee (2007)
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represented lead nitrate induced damage in the histology of air-breathing organs (ABO) of Channa striata in their study. Lead acetate induced micronucleus on cells of Carassus auratus
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auratus was also evaluated by Cavas (2008). Osman et al., (2009) established that lead acetate induces histopathological changes in gills, liver, kidney and spleen of Oreochromis niloticus.
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Studies of Mobarak and Sharaf (2010) revealed that lead acetate exposure resulted in various
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histopathological changes in the gills, liver, hepatopancreas, pancreas, stomach and intestine of
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Poecilia latipinna. Ahmed et al., (2011) reported that lead chloride induced DNA damage using alkaline single cell gel electrophoresis in liver tissue of Anabas testudineus. There are evidences of lead induced histopathological changes in liver, gills, intestine, kidneys and spleen of different fish models. In fish, the innate response has been considered an essential component in combating pathogens due to limitations of the adaptive immune system, their poikilothermic nature, their limited repertoire of antibodies and the slow proliferation, maturation and memory of their lymphocytes (Whyte, 2007). IL-1β and TNF-α have been detected in teleost fish species and is involved in the regulation of immunity (Subramaniam et al., 2002) causing the activation of macrophages, leading to increased respiratory activity, phagocytosis and nitric oxide production (Tafalla et al., 2001). High density fish farming and chemical contaminants in water, 4
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especially chronic exposure to toxic pollutants can lead to decreased immunity. Assays of macrophage function have been used as indicators of fish health (Warinner et al., 1988). Studies had showed that contaminants like lead can affect the phagocytic activity of macrophages in
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Oreochromis niloticus (Omima, 2010).
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However the influence of lead acetate on the non- specific immune system of C. punctatus is still unclear. Thus a better understanding of the mechanisms through which heavy metals influence
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the non-specific immune system is necessary to appreciate the many complex interactions
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between environmental contaminants and the system’s susceptibility to infectious diseases. The aim of the present study was to evaluate the immunotoxicologic effects of lead acetate in
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intestinal macrophages of C. punctatus.
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2.1. Biological material
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2. Materials and methods
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Experiments were performed on Channa punctatus of sizes varying between length 12.08±0.41 and 14.5±0.52 cm and weight 18.07±0.75 and 22.5±0.25 g. Animals were purchased from a local market; upon arrival at the laboratory, the animals were housed in glass aquarium and kept for at least 5-6 days to allow for acclimatization. Only healthy fish that were not diseased, as determined by general appearance (color, skin luster, eyes and behaviour), were used for the studies.
2.2. Exposure After acclimatization, two groups of fish, one for control and the other for lead-treated, were established in a gently aerated semi-static system. Each group consisted of three tanks which 5
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served as triplicates. Each tank housed five fish (n=5). One group was exposed to 9.43 mg/L (2.9 M), 1.02% of 96h LD50 value [925 mg/L] (Devi and Banerjee, 2007) lead acetate solution (prepared in autoclaved distilled water) for 4 days. The other group served as control which was
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kept unexposed to lead acetate. The tank water was changed on a regular basis, the temperature being in the range between 22 and 28ºC, pH 7.2, dissolved O2 6.5 mg/L (90% saturation) and
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salinity ranging from 0.09 to 0.17 mg/l. Fish were fed with minced goat liver and water was
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renewed every 24h. Feeding was allowed to both experimental and control fish throughout the tenure of experiments. All assays were performed in triplicates. The fish were housed, treated
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2.3. Isolation of intestinal macrophages
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and sacrificed following the guidelines of the Institutional Ethics Committee, Assam University.
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The fish were dissected and the whole gut of the fish were isolated, immediately placed in
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Leibovitz medium (L-15) supplemented with heparin (10 IU/ml) and fetal bovine serum (FBS) (2%), and then homogenized in ice cold condition. Cell suspension was then transferred to tubes
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and kept in ice for the settlement of cell debris. The supernatant was layered over Ficoll (45%) and subjected to density- gradient centrifugation (Chung and Secombes, 1998). The band of macrophage-enriched fraction at the interface is collected, washed and resuspended in L-15 medium containing FBS and allowed to adhere on plastic surface. The adherent cells of both exposed and unexposed group were collected and tested for viability at different time intervals as determined by trypan blue dye exclusion technique. Tissue samples showing cell viability higher than 95% were further processed for various analyses.
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2.4. Phagocytosis assay: A volume of 100 µl of cells from both control and exposed groups were allowed to adhere
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separately on glass slides for one hour whereas non-adherent cells were washed out with Dulbecco’s phosphate buffered saline (DPBS) (1X). To the glass slides containing the adhered
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macrophages 10% heat killed Staphylococcus aureus was added and incubated for 3 h at 37ºC which were then washed with DPBS (1X) and dried. The cells were at last fixed in 50%
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methanol, stained with Giemsa, observed under oil immersion microscope and counted for
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number of bacterial cells ingested (Czuprynski et al., 1984). Phagocytosis is expressed as Phagocytic index (%) = (percentage of macrophages containing bacteria) × mean number of
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bacteria per macrophage).
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2.5. Intracellular killing assay:
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Bacteria were incubated with macrophages in L-15 FBS for 20 min at 37oC. After various time
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intervals, samples were plated onto nutrient agar to determine the number of viable intracellular bacteria (Leigh et al., 1986). Intracellular killing was expressed as the percentage decrease in the initial number of viable intracellular bacteria.
2.6. In vitro cell adhesion assay:
Cells were seeded separately for treated and control group in 96 well microtitre plates and allowed to adhere for different times. In time, wells were washed with Hank’s balanced salt solution (HBSS) and then 100 µl of 0.5% crystal violet in 12% neutral formaldehyde, and 10% ethanol was added to each well and incubated for 4 h to fix and stain the cells. Wells were washed and air dried for 30 min. Crystal violet was extracted from the macrophage adhered in 7
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the wells by lysing with 0.1% sodium dodecyl sulphate (SDS) in HBSS. Absorbance was measured spectrophotometrically at 570 nm. Cell adhesion was expressed as increased
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absorbance at 570 nm (Lin et al., 1997).
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2.7. Nitric oxide (NO) release assay:
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Cells (106 cells/ml) were suspended in DPBS and were stimulated with lipopolysaccharide or LPS (100 ng/ml).The cell free supernatants were used for nitric oxide release assay using Griess
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reagent. Readings were taken in a UV spectrophotometer at 550nm. The absorbance was plotted
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on a nitrate standard curve and NO release was expressed as µM (Saggers and Gould, 1989).
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2.8. Myeloperoxidase (MPO) release assay:
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A volume of 100 µl of cells from control and treated group were taken into microcentrifuge
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tubes and stimulated with LPS (100 ng/ml) for 1 hr at 37ºC and centrifuged at 13000 rpm for 10 min. The supernatants thus obtained from both the sets was recovered separately and kept at 20ºC until further use. The cell free supernatant was used for assay of the partial MPO release for both groups. The pellet that recovered from the two groups were lysed in 0.01% SDS and then centrifuged again. The supernatant was recovered as before for total MPO release assay. Subsequently 100 µl of cell free supernatant as well as from lysis of cells were reacted with 100 µl of substrate buffer and kept at 37ºC for 20 min, then the reaction was stopped by adding 100 µl of 2(N) H2SO4 and absorbance was measured at 492 nm (Bos et al., 1990).
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2.9. Ultrastructural analysis of tissue by Scanning Electron Microscope The intestines from treated and untreated groups were excised and rinsed in heparinized saline,
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rinsed in 0.1 M cacodylate buffer at pH 7.5, infiltrated with 2.5% glutaraldehyde for 24 hr fixation at 4ºC, rinsed in buffer, trimmed into 8.0 mm squares and subjected to post-fixation in
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1% OsO4 in 0.1 M cacodylate buffer at pH 7.5 for 2 h and dehydrated through graded acetone. (Spurr, 1969). The mucosal surface of each tissue was mounted on metal stubs, coated with gold
us
using a JFC-1100 (Jeol) ion sputter. Finally, the tissues were scanned with a JSM-6360 (Jeol)
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Scanning Electron Microscope at the Sophisticated Analytical Instrument Facility (SAIF),
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NEHU, Shillong, Meghalaya.
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2.10. Preparation of samples for cytokine analysis
b) From serum
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a) From cell supernatant (in vitro)
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a) Isolated cells were treated with LPS (5 ng/ml) with or without metal treatment. The cells were lysed and any particulate material was removed by centrifugation, supernatant collected and assayed immediately.
b) Peripheral blood was obtained from treated and untreated fish by heart puncture into 1.5 ml microcentrifuge tubes. After cooling overnight at 4ºC, serum was collected after centrifugation and used for cytokine analysis. 2.11. Cytokine analysis: The 96 well microtitre plates (Iwaki Glass, Japan) were coated with 100 µl of fish serum or cell supernatant diluted 10 times in phosphate buffer saline (PBS) and incubated in a moist chamber 9
Page 9 of 37
overnight at 4ºC. TNF- α rabbit polyclonal antibody was used as the primary antibody (Sigma, USA). Vectastain ABC- PO kit (Vector Lab., USA) was applied for ELISA and peroxidase ABTS substrate kit (Vector Lab., USA) was used for coloration. The plate was read at 490 nm on
ip t
a microplate reader (BioRad). For the blank, physiological saline was used instead of serum.
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Fish sera from untreated groups served as control. All assays were performed in triplicate.
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2.12. Statistical analysis
The data were expressed as mean±S.E.M. Data were analyzed using Student’s t-test (two-sample
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assuming unequal variances) for determining the significant change over control values. The
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significance level was set at P<0.05.
3. Results
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Staphylococcus aureus
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3.1. Effect of lead acetate on phagocytic capacity of fish intestinal macrophages against
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Phagocytic index of the control group was found to be 21636.66±5.96 while that of lead treated group was found to be 10821.62±4.14 (P<0.01). [Fig:1] 3.2. Effect of lead acetate on intracellular killing capacity of fish intestinal macrophages On giving bacterial challenge (with S. aureus) to intestinal macrophages of both control and lead treated groups, bacterial load showed significantly higher concentration of viable bacteria at an interval of 15 min (P<0.004), 30 min (P<0.005), 45 min (P<0.002) and 50 min (P<0.007) in the heavy metal treated group as compared to control (Fig:2). 3.3. Effect of lead acetate on cell adhesion property of fish intestinal macrophage. 10
Page 10 of 37
Cell adherence was expressed as the decreased absorbance at 570 nm. After 30 min incubation, absorbance in lead-treated group was found to be 0.07±0.002 from 0.15±0.002 of control group (P<0.04). After 60 min incubation, absorbance in lead-treated group was found to be 0.13±0.002
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from 0.21±0.002 of control group (P<0.02) (Fig:3).
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3.4. Effect of lead acetate on nitric oxide release in fish intestinal macrophages
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NO released from lead-treated+ LPS stimulated intestinal macrophages was found to be
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(4.61±0.197) µM whereas that in control group was (50.94±0.94) µM (P<0.00005) (Fig:4). 3.5. Effect of lead acetate on myeloperoxidase release in fish intestinal macrophages
M
Myeloperoxidase (MPO) release was studied and was found to decrease from (43.51±1.2) % in
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control to (24.42±1.6) % (P<0.02) in lead-treated group (Fig:5).
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3.6. Ultramicroscopic analysis of intestinal tissue by Scanning Electron Microscope
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The present results showed severe damage in epithelium, disarrangement and fragmentation of mucosal foldings and secretion of mucus [Fig: 6(c) and 6(d)] upon lead exposure. Untreated group showed normal epithelium with prominent mucosal foldings [Fig: 6(a) and 6(b)]. 3.8. Effect of lead acetate on TNF- α release from fish intestinal macrophage lysate TNF-α release in intestinal macrophages was studied and was found to decrease from 157 ± 1.28 pg/ml in control to 101 ± 1.14 pg/ml (P<0.02) in lead treated group respectively (Fig:7). 3.7. Effect of lead acetate on serum TNF- α levels
11
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TNF-α release was studied and was found to decrease from 218 ± 1.25 pg/ml in control to 155 ± 1.34 pg/ml (P<0.02) in lead treated group (Fig:8).
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4. Discussion Chronic lead exposure can affect proliferation of white blood lymphocytes. Macrophages play a
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critical role in body’s defense system by eliminating microorganisms from infected tissues
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(Gonsebatt et al., 1992). Phagocytosis by fish cells has been evaluated, both in vivo and in vitro, using different test particles (Estaban et al., 1998; Suzuki, 1986), including yeast cells (Bayne,
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1986; Hyder, 1983; Saggers and Gould, 1989). Macrophages recognize and engulf bacteria into phagosomes, which subsequently acidify. These phagosomes mature into phagolysosomes upon
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vesicle-mediated delivery of various antimicrobial effectors (Garin et al., 2001). From the
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phagocytic index of the present findings it is observed that intestinal macrophages from the
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control group phagocytose S. aureus at a higher rate, which is reduced upon lead exposure. This suggests that heavy metal exposed macrophages fail to perform their normal functions, thereby
Ac ce p
allowing foreign particles or pathogens to gain access to the body which is clearly depicted in the results of the present study.
Professional phagocytes have evolved and are known to internalize and kill as many antigens as possible. Normally, bacteria within macrophages are rapidly killed and degraded in the phagolysosome with the secretion of enzymes and toxic peroxides, making it difficult to dissect the mechanism of death (Slauch, 2011). The present study clearly illustrates a greater percentage of viable bacteria within the lead treated macrophages as compared to that of the control group
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Page 12 of 37
which suggests that lead limits its intracellular killing activity after internalization of the pathogen.
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Chemotaxis is the process by which phagocytic cells are attracted by various molecules like leucocyte specific cell adhesion molecules (CAMs) and migrate to the sites of inflammation,
cr
causing tissue damage or immune reactions. Adherence of antigen to the macrophage cell
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membrane is a vital step in phagocytosis. Complex antigens, such as whole bacterial cells or viral particles, tend to adhere well and are readily phagocytosed (May and Machesky, 2001;
an
Aderem, 2002; Underhill and Ozinsky, 2002). However, this property of cell adherence is significantly inhibited upon lead exposure as it is evident from a fall in absorbance at 570 nm
M
with respect to control. Decrease in in vitro cell adhesion in lead intoxicated group could be
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primarily due to decreased expression of CAMs on the surface of macrophage cells.
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In recent years, NO has been shown to be a very important molecule in regulating immune functions as well as having a direct antimicrobial effect (Gunasegaran et al., 1993; Liew et al.,
Ac ce p
1990). It exhibits a wide range of important functions in vivo, acting as a releasing factor mediating vasodilation, as a neuronal messenger molecule, and as a major regulatory molecule and principal cytotoxic mediator of the immune system (Beckman and Koppenol, 1996; Dimmeler and Zeiher, 1997; Langston et al., 2001). Reactive nitrogen species (RNS), a family of antimicrobial molecules derived from nitric oxide (NO), are produced via the enzymatic activity of inducible nitric oxide synthase 2 (NOS2) (Fang, 2004; Nathan and Shiloh, 2000). The present results illustrate that the amount of NO production by the lead exposed group is strikingly less as compared to the control group. This finding may probably signify suppression of NOS2 expression and subsequent decrease in antimicrobial activity of macrophage.
13
Page 13 of 37
Microbicidals like MPO produced by macrophages are known to play an important role in cellular defenses against various bacterial infections. MPO can generate oxidants from H2O2 and a range of co-substrates, most notably chlorine (Foote et al., 1983) and nitrite (Eiserich, 1998).
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HOCl that is produced by MPO is strongly bactericidal (Klebanoff, 1967) and markedly increases the antibacterial potency of ROS (Rosen and Klebanoff, 1979; Rosen et al., 1990). This
cr
might indicate that MPO is important in host defense in fish. Insufficient release of MPO from
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lead treated macrophages may indicate that its bactericidal potency has been suppressed resulting
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in poor host defense mechanism.
Heavy metals not only deteriorate the physico-chemical equilibrium of the aquatic body, but also
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disrupt the food web and, bring about morphological, physiological and cytogenetic changes in
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the aquatic inhabitants (Yadav and Trivedi, 2009). The functions of teleostean digestive tract
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include digestion, nutrients absorption, hormone secretion, immune protection and water and salt transfers for hydro mineral homeostasis. It regulates energy and material exchange between the
Ac ce p
environment and the internal medium (Khojasteh, 2012). Thus knowledge of the intestinal ultrastructure and morphology is essential for understanding the related functional mechanisms. This study aims to investigate whether heavy metals like lead could induce morphological alterations in fish gut or not. Scanning Electron Microscopy (SEM) is an important tool that reveals information on the surface ultrastucture and morphological changes that may occur in biological tissues. Lead induced severe damages in intestinal epithelium, disarrangement and fragmentation of mucosal foldings, as observed in the present study, cause impairment of tissue organs which in turn has deleterious effects on its immune defense system. These results significantly correlate with the altered non-specific immune functions of the macrophages.
14
Page 14 of 37
Furthermore, lead induced suppression of TNF-α activity, as observed in the present study, may also contribute to the above results.
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Macrophages play an essential role for the initiation and activation of the innate immune system, by recognizing and releasing a large number of cytokines such as tumor necrosis factor- a (TNF-
cr
α), interleukin-1 (IL-1) and IL-6 (Nathan, 1987). The existence of TNF-α like protein in fish has
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been already reported by several authors. Zelikoff et al. (1990) reported the presence of TNF- α in macrophages of rainbow trout, Onchorhynchus mykiss. Hardie et al. (1994) have found that
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rainbow trout lymphocytes and macrophages can functionally respond to human recombinant TNF-α, which suggests that fish leucocytes may possess a specific TNF-α receptor that might
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have been highly conserved in evolution. In addition, TNF-α was one of the cytokines that was
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detected by Ahne, (1993) in the serum of virus infected carp. To determine whether heavy metals
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could alter the secretion of proinflammatory cytokines from macrophages, the release of TNF-α was quantified in lead treated fish serum as compared to control. Our results showed decreased
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TNF-α levels upon metal exposure in serum of lead exposed fish. TNF-α is a proinflammatory cytokine that affects tissue vasculature in inflammation and also induces the acute phase proteins from the liver. In fish, phosphorylcholine reactive protein (PRP), an acute phase protein homologous to C-reactive proteins, is expressed by the action of TNF-α (Szalai et al., 1994).
Serum levels of TNF-α was found to be significantly less in lead treated group than that of control group. This motivated us to investigate the cytokine release from the macrophages of the treated group stimulated with lipopolysaccharides (LPS) under in vitro conditions. Upon LPS
15
Page 15 of 37
stimulation, TNF-α levels in the macrophage supernatant was found to be significantly less in lead treated group than that of control group.
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Lipopolysaccharides (LPS) upregulate proinflammatory cytokines in macrophages, partly through a NF-κB-dependent process. The upregulation of secreted inflammatory cytokines by
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macrophages occurs upon the selective recognition and interaction of the pathogen-associated
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molecular patterns (PAMPs) that are conserved in bacterial species (e.g., lipopeptides, lipopolysaccharides, or flagellin) by the pattern recognition receptors (PRRs) such as the Toll-
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like receptor (TLR) proteins. Perhaps the best characterized example of this microbial recognition by TLRs is the recognition of lipopolysaccharides (LPS) from Gram negative
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bacteria by TLR4. The interaction between LPS and TLR4
signals through Interleukin-1
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Receptor-Associated Kinases (IRAKs), which subsequently signal through Mitogen Activated
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Protein Kinase Kinase 6 (MAPKK6) and the Inhibitor of Nuclear Factor-κB Kinase (IKK). MAPKK6 and IKK activation culminate in the activation of Activator Protein-1 (AP-1) and
Ac ce p
Nuclear Factor-κB (NF-κB) transcription factors, respectively, resulting in the transcriptional upregulation of several proinflammatory cytokines like TNF-α (Chang et al., 2012).
Our findings help in establishing the fact that lead acetate render the fish to an immunocompromised and anti-inflammatory state. Cytokine expression thus becomes downregulated, probably by the action of the heavy metals at multiple levels viz., PAMP recognition, receptor dysfunction and/or by affecting the cell-signalling network.
16
Page 16 of 37
Thus, in the present study, we have examined some of the cellular activities of intestinal macrophages isolated from lead acetate exposed fish, like cell adhesion, phagocytosis and intracellular killing of bacteria. These have been found to be directly related to the cellular
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morphology of the macrophages. Prolonged exposure to heavy metals alters the morphology and diminishes the functional capacities of intestinal macrophages, which can also be correlated, to
cr
their lowered killing capacity in lead exposed fish. In immune cells, which are rapidly
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proliferating and differentiating in response to bacterial challenge, both immune mechanism and cellular activation are critically important. Inhibition of these functions may result in similar
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degree of toxicity. The results in this study provide a baseline for subsequent studies on the role of inflammatory cells in the pathogenesis of bacterial insults during heavy metal intoxication.
M
Since cell adherence was significantly decreased in that of lead exposed cells, it can be suggested
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that in vivo heavy metal intoxication may somehow change the shape, alter membrane signals
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and functionally inactivate the cells so that they migrate at a slower rate. Macrophages contain on their surfaces and also secrete proteolytic enzymes, active at the tissue pH that may be
Ac ce p
important in their ability to migrate in vivo. Numerous substances generated during inflammation, like cytokines and chemokines, have the capacity to enhance the speed of macrophage and orient the movement in the direction of an increased concentration gradient of the agent. Lead exposure may lead to alteration in the expression of cell adhesion molecules on the macrophage membrane as well as the proinflammatory cytokines within the macrophage milieu which further alters the shape and orientation of macrophages so that they migrate slowly and adhere to and engulf bacteria poorly. Reduced adherence of such phagocytic cells to vascular and capillary endothelial cell lining may go without any effective phagocytosis and intracellular killing of the pathogens.
17
Page 17 of 37
Similarly, for initiation of phagocytosis, contact between particulate antigen and phagocytic cell is important. Morphologic alteration of the (lead exposed) macrophages exposed to lead to both reduced cell adhesion as well as phagocytosis. Diminished rate of bacterial clearance from lead
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exposed fish reveals that bacteria may intracellularly survive for a prolonged period indicating susceptibility to severe opportunistic infectious diseases. Rate of bacterial clearance by tissue
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fixed macrophages recovered (isolated) from lead exposed fish may be related to pathogenesis of
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bacteria. The intracellular milieu provides slow growing organisms with an unoccupied niche where microbial competition is relaxed or non-existent. Additionally, an intracellular lifestyle is
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thought to shelter organisms from immune surveillance. However, there is little evidence to support this idea regarding S. aureus infection during lead intoxication. A variation in
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morphology and functional activities of macrophages including enzyme release suggests that the
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immune defense systems of C. punctata intestine are compromised by lead exposure at low
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concentrations, probably by compromising the molecular cross-talk between signaling molecules. Suppression of proinflammatory cytokine release (TNF and IL-1b) implicates the
Ac ce p
involvement of MAPK cascades and downregulation of NFkB genes.
A number of antimicrobial and cytotoxic substances produced by activated macrophages can destroy phagocytized microorganisms. When macrophages are activated with bacterial cell wall lipopolysaccharide (LPS), they begin to express high levels of myeloperoxidase and nitric oxide synthase enzymes as well as the proinflammatory cytokines. NO itself has antimicrobial activity and it can also combine with superoxide anion and peroxide radicals to yield other potent antimicrobial substances. In the present study, it was found that both MPO and NO release significantly decreases in the lead challenged groups as compared to that of control as does the
18
Page 18 of 37
level of the cytokines TNF and IL-1b. So, it can be suggested that lead exposed groups in the face of reduced MPO and NO secretion, are more prone to infections and cannot phagocytose efficiently and as a result, cannot clear the invading microorganism which may lead to diseased
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state upon bacterial invasion. Suppression of proinflammatory cytokine release (TNF and IL-1b) implicates the involvement of MAPK cascades and downregulation of NFkB genes that trigger
us
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these morphological, ultrastructural and functional alterations of the intestinal macrophages.
Conclusively, lead has a deleterious effect on the immune functions of fish, namely on its gut-
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associated lymphoid tissue (GALT). Macrophages constitute the first line of defense against antigenic insults in an organism. A variation in functional activities of macrophages reveals that
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the non- specific immune defense system of C. punctata intestine is compromised by lead
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exposure at low concentrations. The exposure to sub-lethal concentration of lead acetate caused
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suppression of the parameters (phagocytic activity, intracellular killing, cell adhesion, nitric oxide release, myeloperoxidase release and cytokine release) measured in the intestinal
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macrophage of C. punctatus, probably by compromising the molecular cross-talk between signaling molecules. Contamination of water bodies by numerous chemicals and non-essential elements, such as heavy metals like lead, is an unfortunate byproduct of a complex, industrialized, high-tech society. These results also suggest that immunological profiles of fish under heavy metal stress may serve as important bio-indicators in the monitoring of aquatic ecosystems.
Conflict of Interest The authors do not have any conflict of interest in the present work. 19
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Figure Legends:
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Fig:1. Phagocytic activity in intestinal macrophages of fish treated with lead. Values are
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expressed as mean±S.E.M., significant difference with the control group (P<0.01).
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Fig:2. Intracellular killing capacity of control and lead exposed fish intestinal macrophages.
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Values are expressed as mean±S.E.M., significant difference with the control group (P<0.004). Fig:3. Cell adhesion capacity of control and lead exposed fish intestinal macrophages. Values are expressed as mean±S.E.M., significant difference with the control group at 30 minutes (P<0.04) and 60 minutes (P<0.02).
Fig:4. NO released from control and lead exposed fish intestinal macrophages. Values are expressed as mean±S.E.M., significant difference with the control group (P<0.00005). Fig:5. MPO released from control and lead treated fish intestinal macrophages. Values are expressed as mean±S.E.M., significant difference with the control group (P<0.02).
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Fig: 6. Ultramicroscopic changes in (a), (b) control and (c), (d) lead treated fish intestinal tissue. Fig: 7. TNF-α released from control and lead treated fish serum. Values are expressed as mean ±
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S.E.M., significant difference with the control group (P<0.02). Fig: 8. TNF-α released from control and lead treated fish intestinal macrophage lysate. Values
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are expressed as mean ± S.E.M., significant difference with the control group (P<0.02).
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(AQTOX-D-13-00340R1) Lead toxicity on non-specific immune mechanisms of freshwater fish Channa punctatus
Highlights:
Study of lead toxicity in intestinal macrophages of freshwater fish Channa punctatus.
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Lead alters bacterial phagocytosis, intracellular killing and cell adhesion.
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It also inhibits release of antimicrobials like nitric oxide and myeloperoxidase.
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Decreased TNF-α upon lead exposure implicates both MAPK and NFκβ signalling.
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Thus, lead immunocompromises C. punctatus rendering it susceptible to pathogens.
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