Rishikesh K Tiwari and Ravi S
Abstract
Pesticides contributed greatly to pest control and agricultural output whose remarkable proportion reaches to aquatic system by runoff. This results in contamination of water bodies and causes serious health problems to non- target organisms including fish. It also affects the human health by biomagnifications. Therefore, present study was carried out to investigate the sub-lethal effect of chlorpyrifos (organophosphate) cypermethrin (pyrethroid) and their combination to explore their impact on stress parameters such as oxidative stress enzymes (Super Oxide Dismutase; SOD), (Catalase; CAT), (Glutathione-S-Transferase; GST) and antioxidant indices such as (Glutathione Reduced; GSH) and (Lipid peroxidation; LPO) in a stinging catfish Heteropneustes fossiis. Fishes were procured, acclimatised under laboratory conditions and were fed with commercially available fish pellets ad libitum. To study the effect of chlorpyrifos, cypermethrin and their combination, fishes were exposed to 5% & 10% of their LC50 doses up to 96h. After exposure, the fishes were sacrificed and the tissues (brain, liver, kidney, gills and muscles) and blood were collected for estimations. A significant dose-dependent changes (p<0.05) in level of anti-oxidant stress parameters were observed in exposed group as compared to control. This preliminary study reflects the effect of these pesticides which serves as the potential biomarker of pesticides contamination in aquatic system. Abbreviations: AChE – acetylcholine esterase ALA – aminolevulinic acid dehydratase CAT – catalase CYP1A – cytochrome P450, family 1, subfamily A CYP450 – cytochrome P450 DCA– dichloroaniline EROD – ethoxyresorufin-O-deethylase FADH2 – reduced flavin adeninedinucleotide FG – gill cell line FMNH2 – reduced flavin mononucleotide GPX – glutathione peroxidase GR – glutathione reductase GSH – glutathione GSSG – glutathione disulfide GST – glutathione S-transferase G6PD – glucose 6-phosphate dehydrogenase HAHs – halogenated aromatic hydrocarbons HP – hydroperoxide LOOH – fatty acid peroxides LPO – lipid peroxidation L-SH – low molecular mass thiol MDA – malondialdehyde MT – metallothioneins NADH – reduced nicotinamide adenine dinucleotide NADPH – reduced nicotinamide adenine dinucleotide phosphate NADP+ NO – nitric oxide ONOO- – peroxynitrite PAHs – polycyclic aromatic hydrocarbons PMA – phorbol myristate acetate POPs – persistent organic pollutants PUFA – polyunsaturated fatty acids RNS – reactive nitrogen species RO – alkoxyl radical ROO. – peroxyl radical ROS – reactive oxygen species SOD – superoxide dismutase XOD – xanthine oxidase 8-OHdG – 8-hydroxy-2´-deoxyguanosine 8-oxodG – 8- oxo-2´-deoxyguanosine This work is presenting at 19th International Conference on Global Toxicology and Risk Assessment (Global Toxicology 2020) on November 05-06, 2020 at Madrid, Spain OxIDATIVE STRESS The oxidative pressure is a certain part of oxygen consuming life. In the sound high-impact life form, a harmony between the responsive oxygen species(ROS) creation and the framework to shield cells from ROS exists. Command of the ROS creation brings about deformities that may cause cell or life form harms or demise. This awkwardness is alluded to as oxidative pressure (Davies, 1995). The age of ROS emerges by numerous instruments in creature considerably under physiological conditions. There are purported endogenous cell wellsprings of ROS. The most significant of these sources are electron transport chains of mitochondria, endoplasmatic reticulum, the capacity of cytochrome P450 (Di Giulio and Meyer, 2008), the incitation of oxidative chemicals during cata-lysis (Fridovich, 1978; Halliwell and Gutteridge, 1999) and auto-oxidation of the key particles of cell work (Halliwell and Gutteridge, 1999) in creatures, and chloroplasts as an option of mitochondria in plants (Di Giulio and Meyer, 2008). ROS assume additionally a positive job in the creature espe-cially in the phagocytic action of neutrophils and macintosh rophages (Babior, 2000). Upon incitement (for example from opsonized microorganisms), these cells increment O2 utilization up to multiple times resting levels and this is alluded to as "the respiratory burst" (Di Giulio and Meyer, 2008). Procedures which produce oxidative worry in amphibian life forms and warm blooded creatures are comparative. Numerous xenobiotics, for example, pesticides, can prompt the creation of reac-tive oxygen species by a few biochemical instruments, for example, the weakness of film bound electron transport (for example mitochondrial, microsomal electron transport) and ensuing collection of decreased intermediates (Stolze and Nohl, 1994), redox cycling, pho-tosensitization (Di Giulio and Meyer, 2008), help of Fenton response, inactivation of cancer prevention agent chemicals (Kono and Fridovich, 1983) and exhaustion of free extreme scroungers (Winston and Di Giulio, 1991). The activity of ROS brings about lipid peroxidation,protein oxidations, tweak of quality articulation, changes of redox status as cell impacts, and cer tain ailments and untimely maturing as impacts in phase of creature. 2. Antioxidant Defenses THESE components of cell reinforcement guards incorporate chemical frameworks that demonstration to evacuate ROS, low-atomic weight aggravates that directlyscavenge ROS (in creatures, some delivered endoge-nously and others acquired from the eating regimen), and proteins that demonstration to sequester ace oxidants, especially iron and copper (Di Giulio and Meyer, 2008). Cancer prevention agent catalyst frameworks The most significant catalysts for the detoxification of responsive oxygen species in all life forms are superox-ide dismutase (SOD), catalase, glutathione peroxidases (GPXs) and transferases (Di Giulio and Meyer, 2008), xanthine oxidase and glucose 6-phosphate dehydroge-nase (G6PD). Low-sub-atomic weight and different cell reinforcements Glutathione (GSH) exists in two structures, as a diminished GSH and as an oxidized glutathione disulfide (GSSG). Ordinary sound cells contain the diminished structure, GSH, and show GSH: GSSG proportions drawing closer or more prominent than 100:1. Decrease in this proportion can fill in as a marker of oxidative pressure (Di Giulio and Meyer, 2008). Tissue gluta-thione levels are frequently exhausted after transient oxidant exposures yet raised after long haul exposures. Glu-tathione exhaustion sharpens fish, just as vertebrates, to the poisonousness of professional oxidant xenobiotics (Gallagher et al. 1992). Doyotte et al. (1997) and Zhang et al. (2004b) have announced that during a moderate oxidative pressure, the GSH levels can increment as a versatile instrument by methods for an expanded synthesis.Several different biomolecules with a cancer prevention agent func-tion are nutrients, including ascorbic corrosive (nutrient C), tocopherols (nutrient E parts) and carotenoids (α-carotene, β-carotene, beta-cryptoxanthin) which are acquired through the eating regimen in the greater part of the creatures, and the substances with principally other than cell reinforcement work including coenzyme Q, estradiol, bilirubin, lipoic corrosive, uric corrosive (Halliwell and Gutteridge, 1999), metallothioneins (MT) (Coyle et al. 2002) and mela-tonin (Reiter et al. 2008). The new discoveries allude to the cancer prevention agent capability of glucomannan, a water-dissolvable polysaccharide disconnected from yeasts. Bauerova et al. (2008) watched cancer prevention agent impacts of glucomannan detached from Candida utilis. BIomARKERS oF oxIDATIVE STRESS As a presentation it must be said that no single biomarker delicate and explicit enough for oxidative pressure has been distinguished (Di Giulio& Meyer, 2008). By and large, results of oxidative cell or tissue harms and an expanded measure of cancer prevention agent chemicals are watched. Numerous examinations have refered to GSH: GSSG proportions, levels of MT or lipid peroxidation, and exercises of GR, GST and GPX as the most delicate pointers yet these markers have been totally inadmissible in different settings (Di Giulio and Meyer, 2008). Field examines give agent instances of bio-markers of oxidative pressure related with poisons exposures. The significant biomarkers of oxidative pressure incited by pesticides from field examines were recorded by for example Dorval et al. (2005), Eufemia et al. (1997) and Machala et al. (2001). Regularly, biomarkers of oxidative pressure can be isolated into two gatherings: biomarkers with the expectation of complimentary radical harm in natural frameworks and variables of cell reinforcement guards. Biomarkers with the expectation of complimentary radical harm in natural frameworks The utilization of essential and auxiliary results of free extreme harm as biomarkers is helpful for the checking of the oxidative weight of the environ-ment. The most generally utilized biomarkers are results of lipid peroxidation as aldehydes, particularly malon-dialdehyde (MDA), ketones and the assurance of diene conjugation from the polyunsaturated unsaturated fats (Valavanidis et al. 2006). Malondialdehyde is a fundamental optional lipid oxidation result of polyunsaturated unsaturated fats (PUFA). The most significant biomarkers of in vivo oxidativedamage to DNA are results of the particular modifica-tions and hydroxylations of purine and pyrimidine bases and results of harm to the deoxyribosephosphate spine and protein-DNA cross-joins (Valavanidis et al. 2006). The vast majority of the examinations have been centered around the identification of results of the hydroxylation of guanosine, specifically of 8-hydroxy-2'- deoxyguanosine (8-OHdG) or 8-oxo-2'- deoxyguanosine (8-oxodG) and its free base 8-hydroxyguanine (Shigenaga and Ames, 1991). The measure of 8-OHdG in oceanic living beings is considered as a strong biomarker of oxidative worry according to ecological poisons (Rodriguez-Ariza et al.1999; Steinert, 1999; De Almeida et al. 2003). By the hydroxy-lation of thymin two different markers emerge - thymine glycol and thymidine glycol (Valavanidis et al. 2006). The protein oxidation is the last portrayed harm of organic frameworks by free radicals.The estimating is centered principally around carbonyl derivates of proteins, espe-cially the oxidation results of tyrosine (for example dityrosine) (Huggins et al. 1993) and phenylalanine (Valavanidis et al. 2006). Location of γ-glutamyl semialdehyde and 2-amino-adipic semialdehyde is another proof of protein oxidative harm (Valavanidis et al. 2006). Cancer prevention agent protections as biomarkers of oxidative pressure Measuring cancer prevention agent compounds have been utilized as a pointer of the cell reinforcement status of the living being. The compounds that were broke down included superoxide dismutase, catalase, xanthine oxidase (XOD) and glu-tathione redox cycle proteins, glutathione peroxidase, glutathione reductase (GR) and glucose 6-phosphate dehydrogenase. In wild fish progressively explicit markers of oxidative pressure have not developed on account of the assortment of components assume ing job in this issue. They are sex and reproduc-tive condition (Livingstone et al. 1995; McFarland et al. 1999; Meyer et al. 2003; Winzer et al. 2001, 2002a, 2002b), temperature (Heise et al. 2003; Olsen et al. 1999; Parihar and Dubey, 1995; Parihar et al. 1996), diet (George et al. 2000; Hidalgo et al. 2002; Mourente et al. 2000, 2002; Pascual et al. 2003), disintegrated oxygen (Cooper et al. 2002; Hermes-Lima and Zenteno-Savin, 2002; Lush-chak et al. 2001; Ritola et al. 2002b; Ross et al. 2001), saltiness (Kolayli and Keha, 1999; Martinez-Alvarez et al. 2002), occasional impacts (Bacanskas et al. 2004; Ronisz et al. 1999) and physiological or hereditary adjustment to contamination (Elskus et al. 1999; Hahn, 1998; Meyer et al. 2002; Roy et al. 2001). There are no investigations that have detailed huge acceptance of cell reinforcement proteins in fish after ace oxidants introduction (Di Giulio and Meyer, 2008). Now and again, cell reinforcement proteins have been discouraged at the degree of action or articulation after professional oxidants presentation (Fujii and Taniguchi, 1999; Kim and Lee, 1997; Pedrajas et al. 1995; Radi and Matkovics, 1988; Ste-phensen et al. 2002; Zikic et al. 1997). The adjustment to oxidative worry in fish may likewise be a huge factor at times (Bacanskas et al. 2004; McFarland et al. 1999; Meyer et al. 2003). Hasspieler et al. (1994a, b) and Ploch et al. (1999) depicted the capacity of protection from contaminants in fish species because of various antioxi-dant barriers. The fluctuation of cell reinforcement reactions in wild fish might be related with the way that most fish popula-tions are profoundly outbread with a serious extent of hereditary inconstancy (Di Giulio and Meyer, 2008). Moreover the different cell reinforcement isoenzymes, possibly great bio-markers, have been not recognized because of the nonattendance of explicit substrates and antibodies (Di Giulio and Meyer, 2008). An expansion or restraint of the cancer prevention agent compounds levels can rely upon the power and the term of the stressors activation just as the powerlessness of the uncovered fish species (Oruc and Usta, 2007). What's more, the change of the degrees of cancer prevention agent mixes in organ frameworks might be brought about by an alternate concen-tration of xenobiotics in these syste Dichlorvos. MDA content expanded portion depend-ently following dichlorvos introduction in stinging catfish (Heteropneustes fossilis) (Vadhva and Hasan, 1986). The fixation 1–5 mg L-1 of dichlorvos expanded portion conditionally CAT movement in the liver, GSH level in the mind, muscle and heart, SOD in each organ esp. liver, and lipid peroxidaton in the cerebrum in earthy colored bullhead (Ictalurus nebulosus) following 24 hours of the presentation (Hai et al. 1997). Interestingly, similar focuses for a similar treatment period caused GSH decline in the liver and muscle, LPO, GSH, GPX, CAT increment in the mind, SOD and CAT increment in the liver and CAT increment in the kidney of normal carp (Cyprinus carpio) (Hai et al. 1997). It is clear that the oxygen radical impact and the ensuing tissue oxidative harm upon dichlorvos treatment are not as concentrated in like manner carp tissues as in catfish. It might by because of the distinctive ways of life of those two species (Hai et al. 1997). Oxidative worry in anguilla was seen after the introduction to this pesticide (Pena-Llopis et al. 2003). A sublethal con-centration of dichlorvos (0.17 mg L-1, 20 % of the 96h LC50) declined and oxidized strong GSH, declinedthe GSH: GSSG proportion, which is a proof of the procedure ofoxidative pressure. Throb and GR exercises were repressed in the cerebrum. Trichlorfon (TRC). Thomaz et al. (2009) depicted glutathione S-transferase (GST) consumption and hydroperoxide (HP) aggregation after 96 h TRC introduction (0.5 mg L-1) in the core of Nile tilapia (Oreo-chromis niloticus). The heart is the most delicate organ when contrasted with the liver and gills. The cancer prevention agent systems in the gills and liver had the option to expel ROS from TRC incitation, forestalling the expansion of LPO. This may demonstrate a tissue-explicit adjustment to the oxidative conditions after 96 h of TRC expo-sure. The ROS creation and LPO may play a rolein TRC-instigated cytotoxicity. The enzymatic reactions appeared in Nile tilapia after TRC introduction can be identified with the natural cell reinforcement potential and guarded framework introduced by various tissues for what it's worth on account of paper factory emanating presentation (Ahmad et al. 2000). Diazinon. Oruc and Usta (2007) portrayed the expansion of SOD and GPX movement, just as MDA content, in the gill and muscle of Cyprinus carpio after diazinon introduction when contrasted with the control fish. The slight decline of GPX was appeared in the kidney after diazinon introduction. Low exercises of GPX in the kidney of diazinon-uncovered fish exhibit the inef-ficiency of these organs in killing the effect of peroxides (Ahmad et al. 2000). Besides, diazinon introduction essentially expands lipid peroxidation in the muscle of C. carpio. The diazinon-interceded lipid peroxidation is combined with the incited anticholin-ergic action of this pesticide. The harmfulness of diazinon intercedes free radical actuated oxidative cell injury that will in general hoist lipid peroxidation in the gill and muscle tissues of C. carpio. Moreover SOD activ-ity expanded in the tissues of C. carpio after diazinon presentation, and the expansion was increasingly particular in the gill (Oruc and Usta, 2007). This expansion in the SOD action demonstrates the expansion in .O2-creation (Zhang et al.2004a). Oruc and Usta (2007) credited the portion depen-gouge reduction of CAT movement in the muscle of C. carpio to the recorded high SOD movement due to the diazinon-instigated .O2-creation. Diazinon presentation diminishes Na+/K+ - ATPase activ-ity in the muscle and kidney of C. carpio. Two distinct systems can prompt Na+/K+ - ATPase restraint. The first is an immediate impact of ROS and MDA level, and the second a roundabout impact of changes in layer smoothness (Oruc and Usta, 2007). Methylparathion (MP).The analyze portrayed by Isik and Celik (2008) in rainbow trout with the administrator istration of MP and diazinon has demonstrated the huge increment of MDA content in the liver and muscle tissues after 24h and 48h periods, GSH consumption in the liver and gills after similar periods, and GSH increment in the muscle after 48 h introduction to the two pesticides and after 72 h time of diazinon presentation. The cell oxidative worry because of MP can cause the vacillation of SOD action. The abatement in GR action in the tissue is caused halfway by the variety in glutathione concentra-tion in MP and diazinon rewarded cells and somewhat by the moderate GSH recovery. The decrease in GSH content in the tissue can be because of its use to handle the predominant oxidative worry affected by ROS created from MP and diazinon oxidative activation. The reduction in the intracellular degree of GSH and GR movement brought about by MP and diazinon prompts the oxida-tive awkwardness and incites oxidative harm of cells. The examination in Brycon cephalus presented to the sublethal focus (2 mg L−1) of methylparathion for 96 hours was performed by Monteiro et al. (2006). There was watched the huge enlistment of superoxide dismutase, catalase and glutathione S-transferase exercises in the gills, liver and white muscles while the decline in glutathione peroxidase action and increment in LPO values were limited to the gills and white muscle. The expansion of SOD and CAT exercises most likely reacted to the expanded superoxide anions (.O2−) and H2O2 levels (John et al. 2001). The observeddecrease of GPX in the gills and white muscle could berelated to the .O2− creation (Bagnasco et al. 1991) or to the immediate activity of pesticides on the chemical synthe-sister (Bainy et al. 1993). The impressive decrease in the diminished GSH level might be identified with its expanded use and its con-form into oxidized glutathione and a wasteful GSH recovery (Monteiro, 2006). Low hepatic LPO content recommends that this organ is most likely more resis-tant to the oxidative worry because of the enormous range of cancer prevention agent instruments and high nutrient E levels found in the liver of Brycon cephalus (Wilhelm-Filho and Marcon, 1996). The outcomes show that the gills and white muscle are increasingly delicate organs to oxida-tive worry in contrast with the liver. The primary purpose behind these distinctions could be the diverse pace of free extreme age and distinctive cancer prevention agent possibilities in the tissues. The cancer prevention agent arrangement of these tissues isn't as productive as that of the liver. Fenthion. The sublethal fixation (0.45 mg L–1)of fenthion in the cerebrum of Oreochromis niloticus caused the height in GSSG content after 96 h and GPX activ-ity increment after 24 h (Piner et al. 2007). The induc-tion of GPX movement could be identified with the rummage of H2O2 and lipid peroxides by using GSH (Piner et al.2007). Azinphosmethyl. Organization of this pesticide along with 2, 4-dichlorophenoxyacetic corrosive brought about the critical heights of MDA level in the kidney, mind and gill tissue of Nile tilapia (Oreochromis niloti-cus) and regular carp (Cyprinus carpio). In the gill, cerebrum and kidney, CAT movement was higher in O. niloticus than in C. carpio. In the cerebrum and kidney, SOD activ-ity was higher in O. niloticus than in C. carpio. In the gill tissues, movement of SOD and GST didn't show any critical distinction between species. Expanded GST action in the kidney was comparative both in O. niloticus and C. carpio. GPX was expanded more in the gill of C. carpio than that in O. niloticus. The outcomes recommend that the most influenced tissueswere the kidney and gill. Raised GST action may identify with the better assurance against pesticide harmfulness. GPX movement might be expanded because of the expanded professional duction of H2O2 got from .O2− (Oruc et al. 2004).CAT along with the high SOD movement gave anabundant defensive impact (Oruc et al. 2004). The higher CAT movement could mirror the high H2O2 creation from the expanded oxygen utilization (Ritola et al. 2002a). The gill of O. niloticus demonstrated the most noteworthy changes in SOD movement among the tissues (Oruc et al. 2004). The expansion in SOD action could mirror the expanded age of ROS. In this examination, 2,4-D azin-phosmethyl and their joined treatment couldn't invigorate the lipid peroxidation process in the uncovered fish. Low levels or nonattendance of LPO in the tis-sues is a marker of the defensive impacts of antioxidative compounds (Oruc et al. 2004). Azinphosmethyl in the sublethal fixations (2.5 and 5 μg L–1) caused GSH content lessening in the liver and kidney of adolescent rainbow trout after 24 h and 48 h of the introduction and renal and hepatic CAT action decline after 48 h and 96 h of the presentation (Ferrari et al. 2007). The restraint of renal and hepatic CAT is identified with the GSH decline in these organs (Ferrari et al. 2007). Chlorpyriphos (CPF). Mosquito fish (Gambusia affinis) shows the hindrance in exercises of CAT, SOD and GR and the expanded LPO and MDA level after the presentation to 297 μ L–1 (LC50) for 96 h (Kavitha and Ven-kateswara, 2008). These realities propose that the ROS-in-duced harm assumes fundamental job in the poisonous impact of CPF. The steady diminishing in the cancer prevention agent chemicals was because of the unreasonable age of free radicals gener-ated by CPF. The ordinary degrees of cancer prevention agents couldn't extinguish the overabundance of free radicals and in this manner expanded lipid peroxidation. Monocrotophos treatment brought about the decline of CAT action in the liver of asian stinging catfish (Heteropneustes fossilis) (Thomas and Murthy, 1976). Kavitha and Venkateswara (2007) portrayed the enlistment in exercises of CAT, SOD, GR and the expansion of MDA focus in viscera of mosquito fish (Gambusia affinis) after the introduction to 20.49 mg L–1 (LC50) for 96h. It is conceivable that the ROS-prompted harm may beone of the primary poisonous impacts of monocrotophos. a) Carbamates Carbaryl is a bug spray which causes GSH con-tent decline in the liver and kidney of adolescent downpour bow trout (Oncorhynchus mykiss) after 24, 48 Note: This work is presenting at 9th International Conference on Global Toxiology and Risk Assessment on November 05-06, 2020 at Madrid, Spain