Protect Against Carbofuran-Induced Toxicity in Rats

Hindawi Evidence-Based Complementary and Alternative Medicine Volume 2018, Article ID 6254929, 13 pages https://doi.org/10.1155/2018/6254929

Research Article Ganoderma lucidum and Auricularia polytricha Mushrooms Protect against Carbofuran-Induced Toxicity in Rats

Md. Sakib Hossen ,1,2 Md. Maruf Billah Prince,1,3 E. M. Tanvir ,1,3,4 M. Alamgir Zaman Chowdhury,3 Md. Abdur Rahman,3 Fahmida Alam ,5 Sudip Paul ,1 Moumoni Saha,1 Md. Yousuf Ali ,1 Nikhil Chandra Bhoumik,6 Nurul Karim ,1 Siew Hua Gan ,7 and Md. Ibrahim Khalil 1,5

1 Laboratory of Preventive and Integrative Biomedicine, Department of Biochemistry and Molecular Biology, Jahangirnagar University, Savar, Dhaka 1342, Bangladesh 2Department of Biochemistry, Primeasia University, Banani, Dhaka 1213, Bangladesh 3Institute of Food & Radiation Biology, Atomic Energy Research Establishment, Savar, Dhaka 1349, Bangladesh 4School of Pharmacy, Pharmacy Australia Centre of Excellence, Te University of Queensland, Woolloongabba, QLD 4102, Australia 5Human Genome Centre, School of Medical Sciences, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia 6Wazed Miah Science Research Center, Jahangirnagar University, Dhaka 1342, Bangladesh 7School of Pharmacy, Monash University Malaysia, Jalan Lagoon Selatan, 47500 Bandar Sunway, Selangor Darul Ehsan, Malaysia

Correspondence should be addressed to Siew Hua Gan; [email protected] and Md. Ibrahim Khalil; [email protected]

Received 18 August 2017; Revised 31 December 2017; Accepted 8 April 2018; Published 15 May 2018

Academic Editor: Hanan Hagar

Copyright © 2018 Md. Sakib Hossen et al. Tis is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Te current study aimed to investigate the ameliorative efects of two types of mushrooms, Ganoderma lucidum (GL) and Auricularia polytricha (AP), against carbofuran- (CF) induced toxicity in rats. Male Wistar rats (�=42) were divided into six equal groups. Te rats in the negative control group received oral administration of CF at 1 mg/kg with the normal diet for 28 days. Te treatment groups received oral administration of ethanolic extract of GL or AP at 100 mg/kg followed by coadministration of CF at 1 mg/kg with the normal diet for the same experimental period, respectively. In the CF alone treated group, there were signifcant decreases in the erythrocytic and thrombocytic indices but increases in the concentrations of the total leukocytes, including the agranulocytes. A signifcant increase in all of the liver function biomarkers except albumin, in lipid profles except high-density lipoprotein, and in the kidney function markers occurred in the negative control group compared to the rats of the normal control and positive control groups. Te coadministration of mushroom extracts signifcantly ameliorated the toxic efects of the CF. Te GL mushroom extract was more efcacious than that of the AP mushroom, possibly due to the presence of high levels of phenolic compounds and other antioxidants in the GL mushroom.

1. Introduction fsh [2]. Some studies proposed that CF has signifcant harmful efects on the liver and kidney functions as well Carbofuran (2,3-dihydro-2,2-dimethyl-7-benzofuranyl-N as on certain blood parameters of rats [3–5]. Oxidative methyl carbamate; CF) is one of the carbamate pesticides stress has been proposed as an alternative mechanism of CF commonly used to combat insects, mites, and nematodes toxicity in certain animal tissues, occurring via impairment in the soil for fruits, vegetables, and forest crops [1]. Due to of mitochondrial respiratory system that leads to increased the widespread agricultural and household use of this agent, generation of free radicals through lipid peroxidation of cell the contamination of food, water, soil, and air has become membrane which play signifcant roles in the pathogenesis a serious concern based on its inevitable consequences of of a number of complications [6, 7]. Mitigating the oxidative adverse health efects in humans, animals, wildlife, and stress induced by the carbamate pesticides, the exogenous 2 Evidence-Based Complementary and Alternative Medicine supply of antioxidants can improve the capacity of the tissue Germany) at 150 rpm. Tereafer, the extracts were fltered to cope with the high antioxidant demands [8]. through Whatman no. 1 flter paper (Hangzhou Xlnhua Paper Mushrooms are frequently used in traditional Chinese Industry Co. Ltd., China), followed by evaporation using medicine as well as being commonly used as a food item a rotary evaporator (R-215, BUCHI, Switzerland) under a ∘ [9]. Currently, many species of medicinal mushrooms are reduced pressure (100 psi) at a controlled temperature (40 C) commercially available in many countries. Among these to remove any residual solvent. Te samples were then stored ∘ mushrooms, Ganoderma lucidum (GL) (also known as at −20 Cuntilanalysis. Lingzhi or Reishi) [10] and Auricularia polytricha (AP) (also known as cloud ear fungus or jelly ear fungus, a species 2.3. HPLC Analysis of Phenolic Compounds. From the dry that is closely related to Auricularia auricula-judae)arewell extracts, free phenolic compounds were detected following known for their medicinal properties. GL is an oriental previous methods [17, 18] with slight modifcation. Briefy, the fungus with a large, dark glossy exterior and a woody texture extracts (100 mg) were dissolved in 10 ml acidifed water (pH [10].Ithasbeenshowntoproduceantitumorcompounds, 2.0) and extracted with 10 ml methanol. Te organic layer was hypoglycemic polysaccharides, immunomodulatory proteins collected for centrifugation at 6000 rpm for 10 min followed (LZ-8), and many bioactive oxygenated triterpenoids with by fltration through a 0.45 �m syringe flter (Sartorius AG, a very broad spectrum of biological activities and phar- Germany).Tefltrate(5mL)wasthenpassedthrougha macological functions [11]. On the other hand, AP is an 0.20 �m nylon membrane flter (Sigma, USA) and the 20 �L ear-like brown mushroom that grows on wood and its of fltrate was loaded on the HPLC system (SPD-20AV, serial medicinal properties may be contributed by its non-starch number: L20144701414AE, Shimadzu Corporation, Kyoto, polysaccharides, including three D-glucans and two acidic Japan) equipped with a UV detector (SPD-20AV, serial heteropolysaccharides [12]. GL and AP are rich in vitamin B number: L20144701414AE, Shimadzu Corporation, Kyoto, complex, including ribofavin, folate, thiamine, pantothenic Japan). A Luna phenomenex, C18 100A (150 × 4.60 mm, acid, and niacin, and also contain several minerals, such as 5 �m), HPLC column was used. A linear gradient at a fow selenium, potassium, copper, iron, and phosphorus which are rate of 0.5 ml/min was used and the total analytical time was very essential for the maintenance of liver enzyme functions approximately 35 min. Te binary mobile phase consisted of andhelptodetoxifysomecarcinogeniccompoundsinthe solvent A (ultrapure water with 0.1% phosphoric acid) and body [13]. In fact, modern pharmacological and clinical solvent B (pure methanol with 0.1% phosphoric acid). Elution investigations have demonstrated that GL and AP contain from the column was achieved with the following gradient: active constituents with numerous pharmacological efects 0 to 10 min of solvent B, increased from 35% to 55%; 10 to including antitumor, immunomodulatory, cardiovascular, 25minofsolventB,increasedto62%;25to30minofsolvent respiratory, and antihepatotoxic functions [14, 15]. Previous B, increased to 85%, and the fnal composition was kept data have confrmed that the two mushroom species contain constant up to 35 min. Te detection wavelength was fxed high amounts of naturally occurring antioxidants, including between 200 and 450 nm, with specifc monitoring conducted ascorbic acid, tocopherol, and phenolic compounds with at 265 nm. Te identifcation of phenolic and favonoid robust radical scavenging and antioxidant activities [16]. compounds was performed by comparing the retention times Tus, it is hypothesized that GL and AP mushrooms can oftheanalyteswiththereferencestandards.Phenolicsinclud- mitigate oxidative stress and other toxicities induced by CF. ing tannic, gallic, pyrogallol, vanillic, benzoic, and trans- Terefore, the aim of this study was (1) to identify the active cinnamic acids as well as favonoids (including catechin, phenolic compounds in these mushrooms, (2) to determine naringin, rutin, and quercetin) were purchased from Sigma- the efects of CF on hematological and biochemical changes Aldrich (St. Louis, Missouri, USA) and were used as reference in rats, and (3) to determine the ameliorative efects of the GL standards. All solvents were of HPLC grade. and AP mushrooms against CF-induced toxicity. 2.4. Animals. Male Wistar rats with body weights (BW) in 2. Materials and Method the range of 150–155 g were used for the experiment. Te rats were obtained from Department of Pharmacy, Jahangir- 2.1. Pesticide and Mushroom Collection. CF (25 g) was pur- nagar University, and were maintained at a constant room ∘ chased from Shetu Corporation Limited, Bangladesh, in a temperature (23 ± 2 C) in an environment with humidity powdered form (C12H15NO3, purity 98%) and was subse- ∘ ranging from 40% to 70%. Te rats were housed in clean quently stored at 4 C. Te GL and AP mushrooms were plastic cages and received a natural 12-h day-night cycle. collected in powdered form (dry samples were crushed into Teywereprovidedwithstandardfoodandwateradlibitum. a fne powder using a household blender) from the Mush- Te experiments were conducted according to the ethical room Development and Extension Centre, Savar, Dhaka, guidelines as approved by the Bangladesh Association for Bangladesh, in summer following authentication of the Laboratory Animal Science. Te experimental protocol was species. approved by the Biosafety, Biosecurity & Ethical Committee of Jahangirnagar University. 2.2. Extract Preparation. Te powdered mushroom samples ∘ were extracted with 95% ethanol in a conical fask at 30 C 2.5. Study Design. Allratswereacclimatedtothelabenviron- for 72 hours using a shaker incubator (KS 4000 ic, IKA, ment for one week prior to the experiment. Te experiment Evidence-Based Complementary and Alternative Medicine 3 was conducted for 28 days using doses for the mushrooms 2.7. Erythrocytic Parameters. Erythrocytic parameters are andCFthatwereselectedbasedonthosereportedinprevious red blood cells (RBC), hemoglobin (HGB), hematocrit studies [7, 19]. Te hypothesized oxidative stress in rat’s tissue (HCT), mean corpuscular volume (MCV), mean corpuscular was reported to occur following 1 mg/kg of CF treatment [7]. hemoglobin (MCH), mean corpuscular hemoglobin concen- A total of 42 rats were randomly divided into 6 groups (�=7 tration (MCHC), red cell distribution width-standard devi- in each group), as follows: ation (RDW SD), and red cell distribution width-coefcient of variation (RDW CV). (1) Normal control: the rats received 0.5 mL olive oil/rat via oral gavage and a normal diets for 28 days. 2.8. Diferential Leukocyte Counts. Diferential leukocyte (2) Positive control-1: the rats received the GL extract counts are white blood cells (WBC), lymphocytes (LYMPH), (100 mg/kgBW/day) dissolved in saline water via oral monocytes (MONO), neutrophils (NEUT), and eosinophils gavage and a normal diet for 28 days. (EO). (3) Positive control-2: the rats received the AP extract (100 mg/kgBW/day) dissolved in saline water via oral 2.9. Trombolytic Indices. Trombolytic indices are platelets gavage and a normal diet for 28 days. (PLT), mean platelet volume (MPV), platelet distribution width (PDW), pro-calcitonin (PCT), and platelet-to-larger- (4) Negative control: the rats received CF (1 mg/kg BW/ cell ratio (P-LCR). day) (1/5 of lethal dose 50%; LD50)dissolvedinolive oil (0.5 mL/rat) via oral gavage and a normal diet for 28 days. 2.10. Evaluation of Biochemical Parameters. Atotalof 12 biochemical parameters including alanine aminotrans- (5) CF + GL: the rats received the GL extract (100 mg/ ferase (ALT), aspartate aminotransferase (AST), alkaline kg BW/day) dissolved in saline water along with CF phosphatase (ALP), lactate dehydrogenase (LDH), albumin (1 mg/kg BW/day) dissolved in olive oil (0.5 mL/rat) (ALB), total bilirubin (TB), total cholesterol (TC), triglyc- viaoralgavageandanormaldietfor28days. eride (TG), high-density lipoprotein cholesterol (HDL-C), (6) CF + AP: the rats received the AP extract (100 mg/kg low-density lipoprotein cholesterol (LDL-C), creatinine, and BW/day) dissolved in saline water along with CF urea were estimated following standard protocols using an (1 mg/kg BW/day) dissolved in olive oil (0.5 mL/rat) automated chemistry analyzer (Dimension EXL with LM viaoralgavageandanormaldietfor28days. Integrated Chemistry System, Siemens Medical Solutions Inc., USA). All administrations were conducted in the morning (between 09:00 and 10:00 AM). During the experimental period, the 2.11. Oxidative Stress Parameter (LPO). Malondialdehyde rats were observed for any abnormal clinical signs and/or (MDA) levels were assayed to determine the lipid per- death; changes in body weight were evaluated on a weekly oxidation (LPO) products in the tissues according to the basis. method described by Ohkawa et al. [20]. MDA, also referred At the end of the experiment, all rats were sacrifced to as thiobarbituric acid-reactive substance (TBARS), was using deep anesthesia with ketamine hydrochloride injection measured at 532 nm and the levels of TBARS were expressed (1 mL/150 g) followed by dissection. Blood samples (5 mL) as nmol of TBARS per mg of protein. Te total protein in were withdrawn from the inferior vena cava for hematolog- heart tissue homogenates was estimated by the Lowry et al. ical and biochemical analyses using a heparinized syringe method [21]. (5 mL). From each rat, the liver, kidneys, heart, brain, and pancreas were immediately removed and were placed on ice for calculation of the fraction of the body weight. 2.12. Histopathological Examination. Te liver and kidney tis- For the hematological analysis, blood samples (1 mL) were sues were dissected and were fxed in 10% formalin. Te fxed directly transferred into EDTA tubes and were stored at tissues were embedded in parafn and were cut into serial ∘ −20 C until analysis. For the biochemical analyses, the blood sections (5 �mthick)usingarotarymicrotome.Eachsection samples (4 mL) were collected into sterile test tubes and was stained with hematoxylin and eosin (H&E). Micro- allowed to clot at ambient temperature for 30 min followed scopic observation was performed using a light microscope by centrifugation at 2,000 rpm for 10 min to separate the (MZ3000 Micros, St Veit/Glan, Austria). Te pathologist who ∘ serum, which was then stored at −20 Cpriortoanaly- performed the histopathological evaluation was blinded to sis. the treatment assignments of the diferent study groups.

2.6. Evaluation of Hematological Parameters. To evaluate 2.13. Statistical Analysis. All analyses were performed in the hematological parameters, the blood samples (1 mL) triplicate. Te data are expressed as the means ± standard were analyzed following standard protocols using an auto- deviation (SD). Te data were analyzed using GRAPHPAD mated hematology analyzer (XS 1000i, Sysmex Corporation PRISM (version 6.00; GraphPad sofware Inc., San Diego, Ltd., Japan). In this experiment, the recorded hematological CA, USA) and SPSS (Statistical Packages for Social Science, parameters were as follows. version 22.0, IBM Corporation, Armonk, New York). 4 Evidence-Based Complementary and Alternative Medicine

Table 1: Phenolic and favonoid compounds detected in Ganoderma lucidum and Auricularia polytricha mushroom species.

Ganoderma lucidum Auricularia polytricha Serial number Standard compound (mg/kg) (mg/kg) Phenolic compounds 1 Tannic acid 1958.31 ND 2 Gallic acid ND 322.32 3 Pyrogallol ND ND 4 Vanillic acid 2780.21 21.59 5 Benzoic acid ND ND 6 Trans-cinnamic acid ND ND 7 Salicylic acid 7.94 ND Flavonoid compounds 8 Catechin 1013.78 ND 9 Naringin ND ND 10 Rutin ND ND 11 Quercetin ND ND ND: not detected.

(2)(3) (7) (9) (11) 400 750 300 500 200 (mV) (mV) 250 100 (1) (8) (10) (4) (5) (6) 0 0 0 5 10 15 20 25 30 35 0 5 10 15 20 25 30 35 (min) (min) (a) (b) 250 (4) 750 (8) (2) 200

500 150 (mV) (mV) 100 250 50 (1) (7) (7) 0 0 0 5 10 15 20 25 30 35 0 5 10 15 20 25 30 35 (min) (min) (c) (d)

Figure 1: Chromatogram of phenolic compound analysis by HPLC. (a) Standard mixture of seven phenolic acids. (b) Standard mixture of four favonoids. (c) Ganoderma lucidum and (d) Auricularia polytricha. Here, (1) tannic acid, (2) gallic acid, (3) pyrogallol, (4) vanillic acid, (5) benzoic acid, (6) trans-cinnamic acid, (7) salicylic acid, (8) catechin, (9) naringin, (10) rutin, and (11) quercetin.

3. Results During the experimental periods, none of the animals in the normal and positive control groups showed any A total of eleven phenolic compounds were investigated in noticeable signs of toxicity. Noticeable toxic signs, such as this study in which seven were phenolic acids and four mild tremors, sof feces (mild diarrhea), depression, and were favonoid compounds (Figure 1 and Table 1). In GL dyspnea, were observed mainly in negative control group. Te mushroom, three phenolic acids including tannic, vanillic, ratsfromtheCF+GLandtheCF+APgroupsshowedmilder salicylic acids and a favonoid, catechin, were detected. On toxic signs including a rough hair coat and some tremors the other hand, AP mushroom contained gallic and vanillic comparedtothoseinthenegativecontrolgroup.Ofthetotal acids. Overall, the concentration of phenolic acids was higher of 42 rats, only 3 rats died (1 from the CF + AP group and in GL than AP mushroom, indicating the high antioxidant 2 from the negative control group) at 2nd and 3rd weeks, properties of the former. respectively. Evidence-Based Complementary and Alternative Medicine 5

Table 2: Changes in body weight for the various groups.

Parameters Normal control Positive control-1 Positive control-2 Negative control CF + GL CF + AP Initial body weight 152.62 ± 18.20 157.57 ± 17.30∗∗∗ 159.28 ± 11.31∗∗∗∗ 147.00 ± 8.18∗∗∗∗ 148.28 ± 11.31∗∗∗∗ 148.42 ± 8.18∗∗∗∗ (g) Final body weight 233.00 ± 23.94 245.85 ± 14.83∗∗∗∗ 235.00 ± 21.63 204.40 ± 3.57∗∗∗∗ 218.83 ± 21.63∗∗∗∗ 211.25 ± 3.57∗∗∗∗ (g) ∗∗ % changes in body 12.75 13.41 12.09 10.33 12.06 11.01 weight (g) ∗∗∗∗ ∗∗∗∗ ∗∗∗∗ ∗∗∗∗ ∗∗∗∗ Total body weight 80.38 88.28 75.72 57.40 70.55 62.83 gain (g) − Mitigating rate (%) - - - 18.65 8.79 Te values are presented as the means ± SD. Mean values with asterisks (∗) along a row denote level of signifcant diferences compared to the normal control group, obtained using a one-way ANOVA followed by Dunnett’s multiple comparisons test.

Table 3: Changes in the absolute and relative organ weights across the groups.

Mean value of organ weight (g) Mean relative organ weight (g/100 g) Groups Liver Kidney Heart Brain Pancreas Liver Kidney Heart Brain Pancreas Normal control 8.00 ± 1.60 1.40 ± 0.24 0.82 ± 0.11 1.62 ± 0.05 0.8 ± 0.24 3.49 0.60 0.35 0.70 0.35 Positive 8.70 ± 0.78 1.37 ± 0.12 0.79 ± 0.05 1.40 ± 0.02 0.9 ± 0.23 3.53 0.55 0.32 0.60 0.39 control-1 ∗ ∗ Positive 9.30 ± 1.02 1.30 ± 0.10 0.82 ± 0.05 1.50 ± 0.01 0.9 ± 0.17 3.71 0.58 0.34 0.67 0.39 control-2 ∗ ∗ Negative control 7.59 ± 1.45 1.18 ± 0.19 0.74 ± 0.09 1.60 ± 0.07 0.6 ± 0.30 3.95 0.57 0.36 0.79 0.29 ∗ ∗ CF + GL 9.41 ± 1.32 1.36 ± 0.23 0.76 ± 0.14 1.00 ± 0.15 0.9 ± 0.24 4.30 0.62 0.34 0.68 0.42 CF + AP 8.41 ± 1.06 1.39 ± 0.18 0.80 ± 0.17 1.50 ± 0.09 0.6 ± 0.27 3.69 0.61 0.35 0.69 0.29 Te results are expressed as the means ± SD, �=7. ∗ denotes � < 0.05 compared to the normal control group (one-way ANOVA/Dunnett).

Te efects of the mushroom extracts and the CF pesticide Te data for the comparisons of the hematological param- on the body weights and relative organ weight changes are eters in the control and treated groups are shown in Figures showninTables2and3,respectively.Consistentprogressive 2,3,and4.Tenegativecontrolratshadsignifcantlylower 6 increases in the body weights of the rats in the normal values for RBC (10 /�L), HGB (g/dL), HCT (%), MCH (pg), andpositivecontrolgroupswereobserved.Terewasalsoa MCHC (g/dL), RDW SD (fL), and RDW CV (%) compared signifcant increase in the fnal body weight (� < 0.05)atthe to normal control (Figure 2). Some hematological parame- 6 study completion with total body weight in normal control ters [such as RBC (10 /�L), HGB (g/dL), HCT (%), MCH (12.75%)andpositivecontrolrats(13.41%forpositivecontrol- (pg), MCHC (g/dL), and RDW CV (%)] were signifcantly 1 and 12.09% for positive control-2). Te negative control decreased in the rats from the positive control-2 group group showed also a progressive increase in their body compared with normal control group but these values were weights over the 28-day period with a signifcant increase signifcantly higher than those of the rats in the negative of 10.33% in their body weights at the termination of the control group (Figure 2). Supplementation of the CF-treated study, which was signifcantly less than that of the normal and rats with the GL and AP mushrooms (CF + GL and CF + AP positive control groups. On the other hand, the CF + GL and groups) reversed these efects and signifcantly ameliorated the CF + AP groups showed progressive increases of 12.06% the changes in hematological parameters towards the normal and 11.01% of their body weights, respectively, over the study levels. On the other hand, the CF alone treated group (nega- period that indicated protection against the toxic efect of CF. tive control) had signifcantly higher total leukocyte (WBC in 3 Te mitigating rates were determined to be 18.65% for CF + 10 /�L) and agranulocyte counts [LYMPH (%) and MONO GL group and 8.79% for CF + AP group. (%)] but signifcantly lower granulocyte counts [specifcally Administration of CF (at 1 mg/kg) showed signifcant NEUT and EO (%)] than the normal control rats. Te rats in adverse efect on the mean relative liver weights [(liver theCF+GLandtheCF+APgroupshadsignifcantlyhigher weight/body weight) × 100] in the negative control group. LYMPH and fewer granulocytes than the normal control rats However, the mean relative liver weight of the CF + GL group (Figure 3). On the other hand, the negative control group rats 3 was signifcantly increased. On the other hand, for the other showed signifcant decreases in the PLT (10 /�L), MPV (fL), organs (such as kidney, heart, brain, and pancreas), there were PDW (fL), PCT (%), and P-LCR (%) values compared to the no signifcant diferences among the groups for the mean normal control group. Tese thrombolytic parameters were relative weight [(organ weight/body weight) × 100]. signifcantly attenuated in the rats of the CF + GL and the CF 6 Evidence-Based Complementary and Alternative Medicine

Table 4: Te efects of the GL and AP mushroom extracts and carbofuran on the serum biochemical markers.

Biochemical Normal control Positive control-1 Positive control-2 Negative control CF + GL CF + AP parameters Liver function biomarkers ∗ ∗∗ ∗∗∗∗ ∗∗∗ ALT (U/L) 48.75 ± 2.12 38.00 ± 2.88 34.57 ± 3.30 77.75 ± 11.08 31.28 ± 3.98 45.00 ± 10.96 ∗∗∗∗ ∗∗∗ ∗ ∗∗∗∗ ∗∗∗ AST (U/L) 100.71 ± 10.11 61.14 ± 8.19 77.42 ± 5.82 126.37 ± 19.90 71.14 ± 9.40 75.42 ± 9.76 ∗∗∗ ALP (U/L) 238.61 ± 18.95 235.00 ± 18.1 234.57 ± 28.23 311.87 ± 32.86 234.28 ± 14.59 254.57 ± 12.68 ∗∗∗ LDH (U/L) 288.37 ± 32.04 193.71 ± 27.24 224.42 ± 14.22 470.87 ± 31.57 228.85 ± 15.32 245.11 ± 13.07 ∗∗∗∗ ∗ TB (mg/dl) 0.10 ± 0.03 0.10 ± 0.04 0.10 ± 1.50 0.26 ± 0.10 0.11 ± 0.06 0.18 ± 0.06 ∗∗∗∗ ∗∗ ∗∗∗∗ ALB (g/L) 10.60 ± 0.91 11.60 ± 0.91 11.00 ± 1.10 9.25 ± 0.88 10.80 ± 0.62 10.34 ± 0.47 Lipid profle biomarkers ∗∗∗ ∗ TC (mg/dL) 54.87 ± 2.79 53.28 ± 2.81 45.85 ± 3.40 61.87 ± 4.51 50.28 ± 4.42 51.14 ± 5.75 ∗∗∗ ∗ ∗ TG (mg/dL) 48.37 ± 5.34 45.14 ± 2.11 34.42 ± 6.34 52.00 ± 9.12 40.57 ± 3.69 39.85 ± 7.15 HDL-C (mg/dL) 41.00 ± 1.85 41.85 ± 2.47 40.28 ± 2.92 38.00 ± 2.38 41.00 ± 4.08 41.28 ± 4.15 ∗ LDL-C (mg/dL) 04.20 ± 1.80 02.40 ± 1.16 01.68 ± 1.37 10.62 ± 5.11 01.17 ± 0.67 01.88 ± 1.18 Kidney function biomarkers ∗∗∗∗ ∗∗∗∗ ∗∗∗ ∗∗ Urea (mmol/L) 6.50 ± 0.33 4.12 ± 0.74 4.66 ± 0.90 8.14 ± 0.53 6.06 ± 0.74 5.35 ± 0.65 Creatinine 58.85 ± 2.68 48.88 ± 3.56∗∗ 50.20 ± 7.24∗∗∗∗ 64.46 ± 4.07 51.98 ± 5.38∗ 58.80 ± 4.30 (mmol/L) Te values are presented as the means ± SD of three determinations for seven animals per group. Mean values with asterisks (∗) along a row denote level of signifcant diferences compared to the normal control group (one-way ANOVA/Dunnett).

80 ∗ + AP groups compared to the negative control group, which indicated that the mushrooms signifcantly ameliorated the ∗ adverse efects of the CF pesticide on the thrombolytic indices ∗ 60 ∗ ∗ (Figure 4). ∗ ∗ Table 4 shows the overall means of the serum biochemical ∗∗ ∗ parameters. Te data indicated that the CF alone treated 40 ∗ ∗ rats had signifcant increases in the liver function biomarker ∗ levels in serum (such as ALT, AST, ALP, LDH, and TB) but ∗∗ ∗ Value of units of Value ∗ ∗ ∗ ∗ a decrease in the ALB level compared to the normal control 20 ∗ ∗∗ ∗ ∗ group. Supplementation of CF-treated rats with the GL and ∗∗ ∗ AP mushrooms, however, reversed these toxic efects and 0 ameliorated the hepatic marker activities to normal levels. In addition, the lipid profle and kidney biomarker L)

/  data indicated that TC, TG, LDL-C, creatinine, and urea 6

10 were signifcantly increased whereas the HDL-C levels were HCT (%) HCT MCV (fL) MCV MCH (pg) MCH

HGB (g/dL) HGB decreasedinthenegativecontrolgroupfollowingCFalone RBC ( MCHC (g/dL) MCHC RDW_SD (fL) RDW_SD RDW_CV (%) RDW_CV administration compared with normal control. However, the Erythrocytic indices levels of all of these markers were signifcantly normalized Normal Control Negative Control following supplementation of the CF-treated rats with the Positive Control-1 CF + GL mushroom extracts (Table 4). Positive Control-2 CF + AP Based on the investigation of oxidative stress biomarkers, therewasasignifcantincreaseinliverandkidneyLPOlevels Figure 2: Efect of carbofuran on the erythrocyte indices and the in the animals treated with CF alone, as evidenced by the mitigatingefectoftheGLandAPmushroomextracts.Tedata increase in MDA levels when compared to the normal control are expressed as the means ± SD of seven animals per group. Mean group. However, both mushrooms tended to confer a protec- ∗ �< values with asterisks ( ) denote level of signifcant diferences ( tive efect because the animals that were cotreated with the 0.05 ) compared to the normal control group (one-way ANOVA/ GL and AP mushroom extract had signifcantly lower MDA Dunnett). Te units for the vertical axis (�) are expressed as follows: 6 levels compared to the negative control group (Figure 5). Tis RBC (10 /�L), HGB (g/dL), HCT (%), MCV (fL), MCH (pg), MCHC fnding also correlated with the histopathological fndings (g/dL), RDW SD (fL), and RDW CV (%). Red blood cells (RBC), hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume whichrevealedthattheliversandkidneysoftheanimals (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular from the normal and positive control groups had a regular hemoglobin concentration (MCHC), red cell distribution width- organization of hepatocytes that radiated from the central standard deviation (RDW SD), and red cell distribution width- vein (CV) to the periphery of the lobule (Figures 6(a), 6(b), coefcient of variation (RDW CV). and 6(c)) and normal morphology of renal parenchyma with Evidence-Based Complementary and Alternative Medicine 7

100 1.5

∗ ∗ ∗∗ 80 ∗

1.0 60 ∗ ∗ ∗ ∗ ∗ ∗ ∗ 40 ∗ ∗ ∗ Value of units of Value ∗ ∗ ∗ ∗ units of Value 0.5 ∗ 20 ∗ ∗ ∗ ∗ ∗ 0 0.0 3 WBC (10 /L) LYMPH (%) NEUT (%) MONO (%) EO (%) Leukocytic indices Leukocytic indices Normal Control Negative Control Normal Control Negative Control Positive Control-1 CF + GL Positive Control-1 CF + GL Positive Control-2 CF + AP Positive Control-2 CF + AP

Figure 3: Efect of carbofuran on the total and diferential leukocyte counts and the mitigating efect of the GL and AP mushroom extracts. Te data are expressed as the means ± SD of seven animals per group. Mean values with asterisks (∗) denote level of signifcant diferences (� < 0.05) compared to the normal control group (one-way ANOVA/Dunnett). On the vertical axis, the units are expressed as follows: 3 WBC (10 /�L), LYMPH (%), MONO (%), NEUT (%), and EO (%). White blood cells (WBC), lymphocytes (LYMPH), monocytes (MONO), neutrophils (NEUT), and eosinophil (EO).

1000 40 ∗ ∗ ∗ ∗ ∗ ∗ 800 ∗ 30 ∗ ∗ ∗ 600 20 ∗ 400 ∗ ∗ ∗ ∗ Value of units of Value Value of units of Value ∗∗ ∗ ∗∗ 10 200

0 0 3 MPV (fL) PDW (fL) PCT (%) P_LCR (%) PLT (10 /L) Trombocytic Indices Trombocytic Indices Normal Control Negative Control Normal Control Negative Control Positive Control-1 CF + GL Positive Control-1 CF + GL Positive Control-2 CF + AP Positive Control-2 CF + AP

Figure 4: Te efects of carbofuran on the thrombolytic indices and the mitigating efects of the GL and AP mushroom extracts. Te data are expressed as the means ± SD of seven animals per group. Mean values with asterisks (∗) denote level of signifcant diferences (� < 0.05) 3 compared to the normal control group (one-way ANOVA/Dunnett). On the vertical axis, the units are expressed as follows: PLT (10 /�L), MPV (fL), PDW (fL), PCT (%), and P LCR (%). Platelets (PLT), mean platelet volume (MPV), platelet distribution width (PDW), pro- calcitonin (PCT), and platelet-larger cell ratio (P LCR).

well-defned glomeruli (GM) and tubules (Figures 7(a), 7(b), the kidney of the animals from negative control showed and 7(c)). In contrast, the liver of the animals from CF alone degeneration of the tubular epithelium, vacuolization, and treated group (negative control) showed some degenerative moderate glomerular necrosis (Figure 7(d)). Te CF + GL and vicissitudes in the organ, including severe disruption of the theCF+APgroups,however,showedameliorationinallof cellular arrangement, degeneration of hepatocytes at the the investigated histopathological features with moderate to peripheral area of the CV, and congestion in the CV associ- mild degree of worsening of hepatonephrocytes seen (Figures ated with infammatory infltrates (Figure 6(d)). Moreover, 6(e), 6(f), 7(e), and 7(f) and Table 5). 8 Evidence-Based Complementary and Alternative Medicine

4. Discussion Te complete blood count (CBC) is one of the most commonly used blood screening tests and indicates an To our knowledge, our study is the frst to report on animal’s or a person’s overall health. In the present study, we the ameliorative efects of orally administered GL and AP examined three types of blood cells including erythrocytes, mushrooms against CF-induced oxidative injuries in the rats’ lymphocytes, and thrombocytes. Te RBC indices are calcu- liver and kidney. GL conferred better protection against CF- lations derived from the CBC that aid in the diagnosis and induced toxicity than AP for most of the parameters suggest- classifcation of anemia. Several previous studies have shown ing its superior antioxidant activities as also indicated by the that organophosphate and organochlorine insecticides can higher concentration of phenolic compounds determined in alter hematological parameters in experimental animals [29– GL as compared to AP. 31]. Our study confrmed that the carbamate group pesticide Among the eleven phenolic compounds, gallic and vanil- CF also altered the hematological parameters signifcantly. lic acids were detected in AP while GL contained tannic, Te present results revealed that exposure to CF was associ- vanillic, and salicylic acids as well as catechin. Among the ated with signifcant decreases in the RBC, HGB, HCT, MCH, four phenolic compounds, vanillic acid was markedly higher MCHC, RDW SD, and RDW CV parameters. A similar which is also supported by the investigations of Kim et al. trend was observed in rabbits treated with cypermethrin [32]. [22]. Te reduction in the HGB content might be related to the Presently, the use of carbamate pesticides exceeds the use decreased size of RBCs or to the impaired biosynthesis of of organophosphate and organochlorine pesticides, on the heme in the bone marrow [33]. In addition, the reduction in basis that the carbamylated enzyme is much more labile and the blood parameters may be attributed to a hyperactivity of reactivates quickly with a rate between minutes and an hour thebonemarrow[34]leadingtotheproductionofredblood [23].However,someofthecarbamates,especiallyCF,are cells with impaired integrity which were easily destroyed in extremely toxic, and overexposure of individuals involved in the circulation. Shakoori et al. [35] reported that a decrease its production, transportation, and use can result in serious in RBC counts is indicative of either excessive damage to adverse health efects [24]. the erythrocytes or inhibition of erythrocyte formation. Our Because changes in rat body weights may provide an study confrmed that CF induced anemia by reducing the indication of CF-induced toxicity, this was the frst param- total RBCs, HGB, and also HCT (the percentage of blood eter investigated. Te oral administration of CF (1 mg/kg) by volume that is occupied by the red cells). Treatment decreased the total body weight gain of the rats in the with CF also signifcantly reduced the MCV, MCH, MCHC, negative control group compared to the normal control RDW SD, and RDW CV values, which indicated that it group. However, the body weights of treatment groups (CF causes the microcytic type of anemia. However, coadminis- + GL and CF + AP) were improved compared to the negative trationofGLandAPmushroomstoCF-treatedratsincreased control group. Te underlying mechanism for the decrease in these values compared to negative control rats, indicating the body weights of the rats treated with CF may be due to the immunostimulatory efects of the extracts. direct cytotoxic efects of pesticides on somatic cells and/or Furthermore, there were also signifcant increases in the indirect actions through the central nervous system, which WBC, LYMPH, and MONO counts during the treatment controls feed and water intake and regulates the endocrine withCF.Tesefndingsareinagreementwiththosereported system [25]. Another plausible underlying mechanism could in sheep that were treated with cypermethrin [36] and in involve the rapid destruction of the cell membranes via lipid rabbits that were treated with CF and glyphosate [37]. In peroxidation, which occurs when a free radical-mediated addition, CF signifcantly reduced the granulocyte counts attack on the membrane lipids is propagated via an auto- (especially NEUT and EO) compared to those of the control catalytic chain of reactions of lipid peroxidation processes rats as also found by Elsharkawy et al. [29]. Increases in in the presence of oxygen [26]. Because the rats from the leukocyte counts may indicate an activation of the animal’s CF+GLandtheCF+APgroupsconsumedGLandAP defense mechanism [32]. It is plausible that the toxic efects mushrooms with the CF, the cytotoxic and oxidative stress of CF weakened the rats’ immune system, which permitted caused by CF were ameliorated and the body weight gain was the acquisition of infections that stimulated a further immune restored (by 18.65% and 8.79%, respectively) compared with response [30]. On the other hand, decreases in the PLT, MPV, the negative control group. Our study also confrmed that oral PDW,PCT, and P-LCR levels were observed in the CF-treated administration of CF at 1 mg/kg body weight for consecutive group compared to the control group. Tese changes were 28 days has signifcant (� < 0.05) adverse efect on the similar to that reported by Kalender et al. [38, 39]. Tese mean relative liver weights, because the liver is the frst organ decreases occurred due to rapid platelet destruction [29] exposed to metabolizing any upcoming metabolites from as indicated by previous reports that numerous xenobiotics gastrointestinal tract following oral ingestion [27]. However, have the ability to block platelet receptor binding or to the CF administration had no signifcant (� < 0.05)efectson change the platelet membrane charge or permeability [40]. the other organs, such as kidney, heart, brain, and pancreas Coadministration with GL and AP mushrooms signifcantly perhaps because of the low bioavailability; that is, the low normalize these leukocyte and platelet counts in CF-treated dose of CF was less likely to cause large scale toxicity through rats compared to negative control rats. the body [28]. Tese molecular mechanisms may also be In this study, the toxic efects of CF on liver function responsible for the clinical signs and symptoms as mentioned were investigated by determining the activities of various in the Results section. enzymes (ALT, AST, ALP, and LDH) and the plasma protein Evidence-Based Complementary and Alternative Medicine 9

Table 5: Semiquantitative scoring of the architectural changes following histopathological examination of the rat’s liver and kidney.

Scoring parameters Normal control Positive control-1 Positive control-2 Negative control CF + GL CF + AP Liver

Degeneration of 11 1 323 hepatocytes Infammatory cell 11 1 213 infltration Vascular 11 1 321 congestion Edema 11 1 222 Mean ± SD 1.00 ± 0.00 1.00 ± 0.00 1.00 ± 0.00 2.50 ± 0.57 1.75 ± 0.50 2.00 ± 0.81 Kidney

Degeneration of 11 1 212 tubular epithelium Vacuolization 11 1 111 Glomerular 11 1 211 necrosis Mean ± SD 1.00 ± 0.00 1.00 ± 0.00 1.00 ± 0.00 1.67 ± 0.57 1.00 ± 0.00 1.33 ± 0.57 Scoring was performed as follows: 1 = minimal (<1%), 2 = slight (1–25%), and 3 = moderate (26–50%).

20 20 ∗ ∗ ∗ 15 15 ∗ ∗ ∗ ∗ 10 10 ∗ ∗

5 5 TBRAS (nmol/mg of protein) of TBRAS (nmol/mg TBRAS (nmol/mg of protein) of TBRAS (nmol/mg

0 0 CF + GL CF + GL CF + AP CF + AP Normal Control Normal Control Negative Control Negative Negative Control Negative Positive Control-1 Positive Control-2 Positive Control-1 Positive Control-2 (a) (b)

Figure 5: Te efect of GL, AP mushroom, and CF on (a) liver and (b) kidney LPO levels in normal and diferent treated rats. Te values are presented as the means ± SD. ∗ denotes � < 0.05 compared to the normal control group (one-way ANOVA/Dunnett).

ALB. Te results showed signifcant increases in the ALT, in the serum [41]. Another important liver biomarker, total AST,ALP,andLDHlevelsintheCF-treatedgroup.Tese bilirubin, can also be increased in the CF-treated group, changes may occur as a result of enzyme leakage from the which may contribute to the onset of periportal necrosis tissuetotheserumsecondarytoreducedlevelsofhighenergy as reported by Cliford and Rees [42]. Cotreatment with phosphate and phosphocreatine. Tese substances are impor- GL and AP mushrooms signifcantly ameliorated ALT, AST, tant for maintaining the permeability, integrity, and other ALP, LDH, TB, and ALB levels which can be attributed by important characteristics of the cell membrane, including thepresenceofphenolicsandfavonoidsinbothmushroom the maintenance of electrophysiology balance and stability extracts which has inhibitory activities on membrane lipid [41]. CF poisoning results in the expenditure of a signifcant peroxidation with good free radical scavenging activities [43, amount of energy as indicated by the reduction of the ATP 44]. levelsby40–60%.Telackofhighenergyphosphatescan Te lipid biomarker assessment showed that TC, TG, and afect the membrane characteristics, which causes enzymes LDL-C were signifcantly (� < 0.05)increasedinthenegative to leak into the tissue thus increasing their concentrations control rats compared to both normal and positive control 10 Evidence-Based Complementary and Alternative Medicine

CV CV

Normal Control Positive Control-1 (a) (b)

CV CV

Positive Control-2 Negative Control (c) (d)

CV CV

CF + GL CF + AP (e) (f)

Figure 6: Photomicrographs of hematoxylin and eosin stained sections of the liver. (a) Normal control rats showing a normal liver with a hepatic lobule and a uniform pattern of polyhedral hepatocytes radiating from the central vein (CV) towards the periphery, (b) positive control-1 rats showing normal histological structure of the central vein (CV), (c) positive control-2 rats with surrounding hexagonal hepatocytes, and (d) negative control rats demonstrating a disrupted arrangement of hepatocytes around the CV and in the lobule (black arrows), degeneration of the hepatocytes at the peripheral area of the CV (green arrows), and congestion of the CV associated with infammatory infltrates (red arrows). (e and f) Groups CF + GL and CF + AP rats show a remarkable degree of preservation of the cellular arrangement with mild to moderate infammatory infltrates and congestion of the CV [magnifcation: 40x]. rats, whereas the HDL-C levels were decreased. Similar trends mainly synthesized in the liver and intestinal cells. It plays an have previously been observed in studies of other pesticides important role in cholesterol efux from tissues, afer which [45]. Te increase in the serum cholesterol levels may be the cholesterol is carried back into the liver for removal as due to increased cholesterol synthesis in the liver [46]. Te bile acids [48]. It has been established that reduced levels of elevation of the serum TC may also be attributed to the HDL-C are associated with a higher risk of coronary artery stimulation of catecholamines, which promote lipolysis and disease [49]. Moreover, in the treatment groups (CF + GL and increase fatty acid production. Te observed elevation in CF + AP), lipid levels were restored when compared to the the total serum cholesterol levels could be due to blockage negative control rats. Tis may occur due to the inhibition of the liver bile ducts, which would cause a decrease or of cholesterol absorption and biosynthesis which may be cessation of the cholesterol secretion into the duodenum. contributed by the activation of the lipase enzymes as well Tis would subsequently lead to cholestasis. Furthermore, astheincreaseintheexcretionofbileacidsduetotheactive the elevation of the serum TG levels has been attributed to compounds present in the mushroom extracts [16, 43]. inhibition of the lipase enzyme activities that target both Treatment with CF caused a signifcant decrease in the the hepatic triglycerides and plasma lipoproteins [47]. In levels of ALB and increases in the urea and creatinine addition, our study also showed that oral treatment with concentrations compared to the control group. Elevated CF induced a signifcant decrease in serum HDL. HDL is plasma levels of urea and creatinine levels are signifcant Evidence-Based Complementary and Alternative Medicine 11

GM GM GM GM

Normal Control Positive Control-1 (a) (b)

GM GM GM

Positive Control-2 Negative Control (c) (d)

GM GM

CF + GL CF + AP (e) (f)

Figure 7: Photomicrographs of hematoxylin and eosin stained sections of the kidney. (a) Normal control rats showing a normal morphology of renal parenchyma with well-defned glomeruli (GM) and tubules; (b and c) positive control-1 and positive control-2 showing a normal appearance of the renal parenchyma; (d) negative control rats demonstrating marked degeneration of the tubular epithelium (black arrows), vacuolization (red arrows), and slight glomerular necrosis; and (e & f) Groups CF + GL and CF + AP rats showing a remarkable degree of morphological preservation with only slight to minimal necrosis of the epithelium [magnifcation: 40x]. markers of renal dysfunction [50]. Low creatinine and urea appears to be the initial stage where the liver and kidney clearance indicate a weakened ability of the kidneys to flter tissue become more susceptible to oxidative damage. How- these waste products from the blood and excrete them in ever, coadministration with the two mushrooms signifcantly the urine. Moreover, elevated blood urea is correlated with reduced the levels of lipid peroxides in CF-exposed rats increased protein catabolism in the body [51]. Pesticide- suggesting its protective efects against oxidative damage. induced increases in the urea level as observed in the present Moreover, our histomorphological observations in the liver studymaybeduetotheefectofCFontheliverfunction and kidney tissues also confrmed the protective efects of because urea is the end product of protein catabolism [52]. GLandAPonCF-inducedtoxicity.Teliversofnegative Tis possibility is supported by the decrease in the levels of control rats showed a moderately disrupted arrangement of the plasma protein ALB. Coadministration of mushrooms the hepatocytes with congestion of the central vein seen. In a extracts (GL and AP mushroom) normalized the ALB, urea, recent study, marked changes in the biochemical parameters and creatinine level compared to negative control rats. oftheliverandageneralizedcongestionoftheportal, LPO generally refers to the oxidative degradation of lipids centralvenous,andsinusoidalpartoftheliverwerealso in which free radicals acquire the electrons from the lipids seen when the rats were treated with CF at 1 mg/kg body in cell membranes, resulting in cell damage. Increased LPO weight for fve weeks [53]. It is plausible that the active 12 Evidence-Based Complementary and Alternative Medicine constituents in GL (including tannic, vanillic, salicylic acids, [8]S.F.Ambali,D.O.Akanbi,M.Shittuetal.,“Chlorpyrifos- and catechin) and AP (including gallic and vanillic acids) induced clinical, haematological and biochemical changes in with antioxidant activities scavenge the free radicals and Swiss albino mice: mitigating efect by co-administration of confer some protection on the liver and kidney tissue. vitamins C and E,” Life Science Journal,vol.7,no.3,pp.37–44, 2010. [9] J. Yang, H. Lin, and J. Mau, “Antioxidant properties of several 5. Conclusion commercial mushrooms,” Food Chemistry,vol.77,no.2,pp. 229–235, 2002. Our study confrmed that the GL and AP mushrooms confer [10]S.Wachtel-Galor,J.Yuen,J.A.Buswell,andI.F.F.Benzie,Gan- signifcant protective efects against CF-induced toxic efects oderma lucidum (Lingzhi or Reishi): A Medicinal Mushroom, on the body weight, hematological, and several biochemical 2001. parameters and that the GL mushroom provided better [11] B. S. Sanodiya, G. S. Takur, R. K. Baghel, G. B. K. S. Prasad, protective efects than the AP mushroom against CF-induced andP.S.Bisen,“Ganoderma lucidum: s potent pharmacological toxicity. macrofungus,” Current Pharmaceutical Biotechnology,vol.10, no. 8, pp. 717–742, 2009. Conflicts of Interest [12]Z.Zhang,W.K.K.Ho,Y.Huang,E.J.Anthony,L.W.Lam,and Z.-Y. Chen, “Hawthorn fruit is hypolipidemic in rabbits fed a Te authors declare that there are no conficts of interest. high cholesterol diet,” Journal of Nutrition,vol.132,no.1,pp.5– 10, 2002. 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