Croton tiglium extract was selected to be incorporated by silver nanoparticles (Ag-NPs) then evaluated against colon cancer induced by azoxymethane (AOM) in rats. Colorectal cancer (CRC) is considered as the most common type of gastrointestinal cancers. Chemotherapy became limited due to the adverse side effects. Therefore, the most effective P53 (TP53) and Adenomatous Polyposis Coli (APC) genes in colonic tissues. Different hematological and biochemical measurements were quantified in addition to markers of oxidative stress. Specific tumor and inflammatory markers were assayed. Colonic tissues were examined histopathologically in addition to immunohistochemistry (IHC). Native proteins and isoenzymes patterns were electrophoretically assayed beside expression of Tumor Protein C. tiglium nano-extract restored these measurements to normalcy. Tumor and inflammatory markers elevated significantly (P≤0.05) in sera of AOM induced colon cancer group in addition to increasing peroxidation products with decline in antioxidant enzymes activities in colon tissues. Nano-extract restored these measurements to normalcy in post-treated group. Histopathological study revealed that nano-extract minimized severity of inflammatory reactions in all nano-extract treated groups and prevented anti-Keratin 20 antibody expression in post-treated group. The lowest similarity index (SI%) values were noticed with electrophoretic protein (SI=71.43%), lipid (SI=0.00%) and calcium (SI=75.00%) moieties of protein patterns, catalase (SI=85.71%), peroxidase (SI=85.71%), α-esterase (SI=50.00%) and β-esterase (SI=50.00%) isoenzymes in colon cancer group. Furthermore, AOM altered the relative quantities of total native bands. The nano-extract prevented the alterations that occurred qualitatively in nano-extract post-treated group and quantitatively in all nano-extract treated groups. Levels of TP53 and APC gene expression increased in AOM injected group and nano-extract restored their levels to normalcy in the post-treated group. It was found that AOM caused significant (P≤0.05) elevation in the hematological and biochemical measurements. C. tiglium nano-extract exhibited ameliorative effect against the biochemical and molecular alterations induced by AOM in nano-extract post-treated group. Colorectal cancer (CRC) is categorized as one of the most common types of gastrointestinal cancers that studied extensively in the world. It is considered as the third highest cause of cancer death . The CRC pathogenesis involves initiation of epithelial cells to a cancerous state with defined precancerous intermediaries by sequential and multistep progression . Aberrant crypt foci are abnormal clusters identified early in the lining of colon and rectum before colorectal polyps as precancerous lesions during colon carcinogenesis .Success of cancer chemotherapy is limited due to the adverse effects induced by the drug, multidrug resistance and off-targets binding . In order to develop new cancer chemotherapy, it is necessary to induce cancer tumors in rats to evaluate the efficacy of the potential anti-cancer drugs. Many carcinogens are able to induce colon tumors in rats but azoxymethane (AOM) has been most widely used . Incidence of colon tumors induced by two or three subcutaneous injections of AOM takes 7-9 months to induce sufficient tumors . When an inflammatory agent is administered via drinking water to the AOM-treated rats, it accelerates the induction process and it was postulated that dextran sodium sulfate (DSS) is the most suitable inflammatory agent which is able to develop number of colon tumors within a short time period .polyposis coli (APC) gene participates in signaling transduction pathway. Mutations in the APC gene play a rate-limiting role in majority of sporadic colorectal cancers and hypermethylation of its promoter is closely related to cancer development . A tumor suppressor protein encoded by tumor suppressor P53 (TP53) gene that regulates expression of the genes involving apoptosis, angiogenesis and genome maintenance. Mutations in TP53 genes appear to occur at relatively late stage of colorectal cancer .Biomarkers such as carcinoembryonic antigen (CEA) and carbohydrate antigen (CA) 19-9 have the characteristics that enable them to detect the progression of CRC and gastrointestinal tumor at earlier stages . Alterations of specific oncogenes and tumor suppressor genes play a role at different stages of carcinogenesis process . Protein encoded by adenomatous Croton tiglium L is one of the largest genera of flowering plants which are widely used in ethnomedicine for the treatment of several cancer diseases . It exhibited high antioxidant activity due to the presence of some other phytochemicals such as ascorbic acid, tocoferol and other pigments . The croton oil isolated from C. tiglium seeds used for treatment of the solid tumors due to presence of 12-O-tetradecanoylphorbol-13-acetate (the major active constituent) that is able to stimulate apoptosis in prostate, breast, colon and lung cancers .The synthetic antioxidants have restrictions for medicinal uses, as they are suspected to be carcinogenic . Recent studies in medicinal plants field aimed to find suitable natural antioxidants for therapeutic purposes. These antioxidants can reduce free radicals induced by tissue injury . C. tiglium extract can cause increase in plasmid DNA strand breaking. This increase depends on the concentration used of the extract. Therefore, the plant extract needs to be tested to determine the required dose before directed to therapeutic purposes . Recent study showed that C. tiglium seeds extracts have no significant alterations in the biochemical measurements such as total protein, albumin and globulins of rats fed with C. tiglium seeds extracts. However, these extracts have significant effect on some haematological indices .It was emphasized that aqueous Most of the biologically active components absorbed slowly due to their high molecular weights which decreases their ability to cross the cellular membrane and hence decreases their efficacy and bioavailability. Therefore, integration of the traditional medicinal plants with nanotechnology can overcome such problems by increase their bioavailability and reduce their toxicity . Development of nano-extracts containing metal nanoparticles (M-NPs) showed great promise to solve their inherent stability problem. Incorporation of M-NPs into polymeric matrices showed valuable properties in many practical applications . The plant extracts with M-NPs increased the total phenolic compounds and exhibited good antioxidant activity at lower concentrations . Therefore, it showed a higher antioxidant and antimicrobial activity as compared to plant extract solely . C. tiglium extract to enhance its antioxidant efficiency as suggested by Aboulthana et al., (2019) then to evaluate nano-extract against colon cancer induced by AOM in rats.From this point of view, the study was designed to develop novel strategies for prevention of colon cancer by mean of green nanotechnology through incorporating Ag-NPs into Silver C. tiglium nano-extract administrationC. tiglium nano-extract was prepared and characterized by advanced confirmatory technique and administrated orally by stomach tube at a dose of 6.5 ml/Kg body weight (b.wt)  .The aqueous silver Induction of colon cancer in ratsC. tiglium nano-extract against colon cancer. Rats were injected by a single injection of AOM at a dose of 20 mg/kg b.wt interperitoneally (i.p.) to induce colon carcinogenesis chemically then rats were given 2% DSS in drinking water for 7 days after one week of AOM injection. Histological examination for injected rats was done from 8th week of AOM injection to detect disease induction . It was found that the disease induced after 12 weeks. The induced rats were consequently used to evaluate efficacy of the prepared silver Experimental design Healthy thirty-six adult male Wistar rats (weighting 120 - 150 g) were housed in cages (six per cage). Rats were provided with water ad libitum and maintained under normal nutritional and environmental conditions at 25 ± 2°C. The experimental procedures were carried out according to the ethical protocol and guidelines approved by the institutional animal care of National Research Centre, Dokki, Giza, Egypt. C. tiglium nano-extract treated group: Rats were fed with normal diet associated with the treatment with C. tiglium nano-extract orally at a dose of 6.5 ml/Kg b.wt for 21 days, Colon cancer induced group: Rats were injected with AOM at a dose of 20 mg/kg i.p., Silver C. tiglium nano-extract pre-treated group: Rats were treated with C. tiglium nano-extract orally at a dose of 6.5 ml/Kg b.wt for 21 days followed by injecting AOM i.p. for another 12 weeks, Silver C. tiglium nano-extract simult.-treated group: Rats treated simultaneously with AOM i.p. and treated orally by C. tiglium nano-extract and Silver C. tiglium nano-extract post-treated group: Rats were injected AOM i.p. for 12 weeks followed by the treatment with C. tiglium nano-extract orally at a dose of 6.5 ml/Kg b.wt for another 21 days.Rats were randomly divided into Control group: Rats were fed with normal diet and received distilled water for 21 days, Silver Collection of blood samples and tissuesRats were anaesthetized and sacrificed by decapitation at end of the experimental period then heparinized blood samples were collected from retro orbital plexus from each rat for the hematological measurements and serum samples were collected for biochemical measurements by centrifuging blood samples for 15 min at 4,000 rpm. In addition, colon tissues were collected then excised and washed in ice-cold saline. All collected colon tissues were autopased and splitted into 2 portions. Portion (I) was immediately fixed in 10% formal saline until the histopathological examination. Portion (II) was immediately washed in physiological saline then homogenized in Tris-HCl buffer (0.01 M and pH 7.4). Aliquots of the clear supernatants were used for measuring the inflammatory and oxidative stress markers and for undergoing the different electrophoretic patterns.Hematological and biochemical measurementsHeparinized blood samples were analyzed on an automatic blood analyzer (ABX Micros 60 manufactured by HORIBA ABX SAS) to quantify hemoglobin (HB), red blood cells (RBCs), hematocrit (HCT), corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), red blood cell distribution width (RDW), mean platelet volume (MPV) and platelet count (PLT), white blood cells (WBCs) and differential blood cells . While serum blood samples were used to estimate all traditional biochemical parameters  and aspartate transaminase (AST), lipid profile  and triglycerides (T.Gs)), renal functions (urea and creatinin) and protein profile ) spectrophotometrically by colorimetric methods following instruction of the commercial kits purchased from Spectrum Diagnostics Egyptian Company for Biotechnology .Tumor markersTumor markers specific for colon cancer including Carcinoembryonic Antigen (CEA) and Carbohydrate Antigen (CA) 19-9 were assayed quantitatively in sera of all groups using Enzyme Linked Immune Sorbent Assay (ELISA) technique.Inflammatory markersMarkers of the inflammatory response specific for colon cancer were quantified through measuring C-reactive protein (C-RP) level and Myeloperoxidase (MPO) activity. C-RP level was determined in serum samples and expressed as mg/L using commercial enzymatic kit . While, MPO activity was determined in supernatant of colon tissue homogenate and expressed by Δ/min/mg protein .The major cellular contents in colon tissues homogenatesThree cellular contents were assayed as following calcium concentration was determined by using fully automated with microprocessor-controlled electrolyte system (Sensa Core’s ST-200 CL) and Lactate dehydrogenase (LDH) activity was done according to Babson and Babson (1973). Finally, Glucose level was measured by enzymatic colorimetric method following instructions of the Kit manufacture .Markers of Oxidative StressAll these markers were assayed by using clear supernatants of colon tissue homogenates. These assays including: i) Products of the peroxidation reactions: Lipid peroxidation product (LPO) was determined and expressed as malondialdehyde (MDA) which is the end product of peroxidation reaction and the result was expressed as (nmol/g wet tissue) . While total protein carbonyl content (TPC) was quantified spectrophotometrically and expressed as nmol of reactive carbonyl compounds per mg protein of tissue . ii) Antioxidant enzymes: Total Antioxidant Capacity (TAC) was determined and expressed as mM/L . While Catalase Activity (CAT) was assayed and expressed as Unit per gram of tissue . Finally, Glutathione Peroxidase activity (GPx) was measured and expressed as Unit per gram of tissue . Statistical Analysis P” value of less than 0.05.All data were statistically analyzed by one-way analysis of variance test (one-way ANOVA) using the Statistical Package for Social Sciences  followed by Bonferoni test. Results were expressed as mean ± standard error (SE). The differences between the groups were considered statistically significant when a “Histopathological ExaminationAfter sacrifice, specimens of colonic tissues were autopsied from different groups then studied histopathologically . All samples were immediately fixed in 10% formal saline for 24 hr and washed in tap water then dehydrated in serial dilutions of alcohol solutions. Tissue fragments were then cleared in xylene and embedded in the paraffin bees wax blocks that were prepared for sectioning at 4 microns thickness by slidge microtome. The tissue sections were collected on glass slides and deparaffinized then stained by hematoxylin and eosin (H and E) stain for the examination under light electric microscope to confirm occurrence of the induction process. The histopathological changes were scored . A rating score between 0 (no damage) and +++  was assigned for carefully investigated section. Also, the induction process was confirmed by the immunohistochemistry (IHC) technique for detection and localization of the anti-Keratin 20 antibody in colon tissue .Electrophoretic AssaysKnown weights of colon tissues (0.2 gm) were freezed rapidly in liquid nitrogen then homogenized in 1 ml water-soluble extraction buffer. The homogenates were centrifuged at 10.000 rpm for 5 min. and the clear supernatants transferred into another tubes. Known volumes of the individual homogenates supernatants were pooled together and used as one sample for each group. Concentration of total protein was determined in all pooled samples . Protein concentration must be equal in all wells during all electrophoretic assays. These assays including:i) Electrophoretic protein pattern: Native proteins were separated electrophoretically through Polyacrylamide Gel Electrophoresis (PAGE) .ii) Electrophoretic lipid moiety of native proteinpattern: Native gels were stained for detecting lipid moiety of native protein with Sudan Black B (SBB).The stained lipoprotein bands appeared as black bands. The Rf, B% and Qty% of lipoprotein bands were determined Subramaniam and Chaubal (1990).iii) Electrophoretic calcium moiety of native protein pattern: Native gels were stained for detecting calcium moiety of native protein using alizarin Red ‘S’. The stained calcium moieties appeared as yellow bands. The Rf, B% and Qty% of the calcium moiety bands were determined .2O2 as substrate then stained.The stained CAT subunits appeared as yellow bands in the gel . While, the stained POX subunits appeared as brown bands in the gel . Finally, Electrophoretic esterase (EST) pattern was carried out to localize in-gel EST activity by incubating gel in conditioning buffer then staining in reaction mixture consisting of α, β-naphthyl acetate  as substrates along with dye coupler Fast Blue RR. The α-naphthyl acetate used as substrate for α-esterases which appeared as brown bands and β-naphthyl acetate used as substrate for β-esterases which appeared as dark pink bands Ahmad et al., (2012).iv) Electrophoretic isoenzymes (zymography): Non-denatured PAGE is used for detecting the enzyme activity through identification of the enzyme subunits. Firstly, electrophoretic catalase pattern (CAT) and Electrophoretic peroxidase (POX) pattern were assayed for native gel after electrophoretic run by incubating with HData AnalysisPAGE plates were photographed and analyzed using Quantity One software (Version 4.6.2). The relative mobility (Rf), band percent (B%) and relative band quantity (Qty%) of the electrophoretically separated bands were determined in addition to the molecular weights (Mwts) which were determined by this program in comparison to marker of standard molecular weights with regularly spaced bands ranging from 6.458 to 195.755 KDa. The similarity index (SI%) and genetic distance (GD%) were calculated . Molecular AssayRNA extraction and cDNA synthesis® . Purity of the RNA yield was accepted in all samples (A260/A280 absorbance ratio of between 1.85 and 2.0). All RNA samples were normalized to1,000 ng/μL, DNase treated  and reverse transcribed into First Strand cDNA using Power cDNA Synthesis Kit (iNTRON Biotechnology) according to manufacturers’ protocols.Total RNA was extracted from colon tissues using easy-spinTM Total RNA Extraction Kit (iNTRON Biotechnology). The RNA concentration and purity were determined using NanoDropPrimer design APCSpecific primers for  andTP53 genes were designed on the bases of the Rattus norvegicus sequence Gene bank accessions NM_012499 and NM_030989 respectively using Primer Quest Tool and synthesized by Macrogen company. The designed primers were used for Real time quantitative PCR (qRT-PCR). For gene expression analysis, Beta actin (Actb) was used as endogenous control and its primers  were designed based on sequence of Gene bank NM_031144.Gene expression using qRT-PCRAPC and TP53 genes were determined in colon tissues of control and different treated groups. A master mix was prepared for each assay containing 10μL of 2X SYBR green PCR mix , 1 ul of 10uM primer sets and nuclease-free water. For each sample, a 25 μL reaction volume of mastermix containing 1 ul of cDNA was prepared. The qPCR was performed by Qiagen’s real-time PCR system (Rotor-Gene Q) using default cycling conditions : 40 cycles of an initial activation step of 95°C for 15 min followed by 95°C for 10 s (melt), 57°C for 15s  and 72°C for 30s (extend). The mean Cycle threshold (Ct) values were calculated and then normalized to Actb using ΔCt method. Changes in relative expression were calculated using the 2-ΔΔCt method .Expression of the Statistical analysisMolecular data were statistically analyzed using the SPSS computer program . Levels of the relative gene expression were compared between control and other treated groups using the non-parametric Mann-Whitney test. Results were reported as the arithmetic mean ± SE, and P value less than 0.05 is considered statistically significant .C. tiglium extract after incorporating Ag-NPs against colon cancer induced by AOM, it was found that AOM caused adverse effects at the hematological and biochemical levels during the induction process.Although the present study was designed to reveal efficiency of Hematological MeasurementsC. tiglium nano-extract caused significant (P≤0.05) decrease in levels of all these measurements in all nano-extract treated groups as compared to AOM induced colon cancer group, it restored these measurements to normal values in post-treated group.As shown in Biochemical Measurements C. tiglium nano-extract treated groups as compared to AOM induced colon cancer group. Furthermore, AOM affected kidney functions through increasing levels of urea and creatinin and decreasing levels of T.P and albumin significantly (P≤0.05). Moreover, all C. tiglium nano-extract treated groups showed significant (P≤0.05) decrease in levels of urea and creatinin significantly (P≤0.05) with restoring T.P and albumin levels to normal values.As reported in Tumor MarkersC. tiglium nano-extract declined levels of those markers significantly (P≤0.05) in all nano-extract treated groups and restored their levels to normalcy in post-treated group.Data illustrated in Inflammation MarkersAs regard to the inflammatory response, data illustrated graphically in C. tiglium nano-extract caused significant (P≤0.05) decrease in levels of inflammatory markers in all nano-extract treated groups as compared to AOM induced colon cancer group, it restored levels of these measurements to normal values in post-treated group.Although the treatment with Major Colonic Contents and Oxidative Stress MarkersAs regard to the assays that were carried out in the colon tissues, glucose and calcium in addition to LDH enzyme are considered as the major cellular contents in colon tissues. AOM caused significant (P≤0.05) decline in these measurements as compared to control group .C. tiglium nano-extract caused significant (P≤0.05) increase in glucose level in all nano-extract treated groups as compared to AOM induced colon cancer group and restored its level to normalcy in post-treated group. On the other hand, it decreased activity of LDH as compared to AOM induced colon cancer group and could not restore its activity to normal value in all nano-extract treated groups. As regard to calcium level, C. tiglium nano-extract caused significant (P≤0.05) increase in its level with respect to AOM induced colon cancer group and restored its level to normalcy in all nano-extract treated groups.Although the treatment with Moreover, AOM caused significant (P≤0.05) decrease in level of the total antioxidant capacity (TAC) and activities of the antioxidant enzymes (CAT and GPx) associated with significant (P≤0.05) elevation in concentrations of the peroxidation products (LPO and TPC) as compared to control group as shown in C. tiglium nano-extract caused significant (P≤0.05) increase in TAC and activities of CAT and GPx associated with lowering concentrations of LPO and TPC in all nano-extract treated groups as compared to AOM induced colon cancer group, it restored these measurements to normal levels in post-treated group.Although the treatment with Histopathological Examination and ImmunohistochemistryC. tiglium nano-extract treated group  as well as anaplasia and dysplasia  in the glandular epithelial structure as hyperchromachia, polarity and pleomorphism .C. tiglium nano-extract minimized severity of the inflammatory responses that is considered as criteria of malignancy and stage of colon cancer especially in the nano-extract post-treated group.In nano-extract pre-treated group, sever lymphoid follicles hyperplasia  was detected in the submucosa and extended to the lamina propria of the mucosa . In nanoC. tiglium nano-extract treated groups. While in the AOM induced colon cancer group, anti-Keratin 20 antibody was expressed severely . Although the treatment with C. tiglium nano-extract could not prevent the presence of anti-Keratin 20 antibody completely in the pre-treated and simult-treated groups, it reduced its expression obviously . While in nano-extract post-treated group, it prevented expression of anti-Keratin 20 antibody completely.On the other hand, the IHC was carried out for detection of the anti-Keratin 20 antibody through localization of the reaction on the tips of the mucosal epithelium. Severity of the immunoreactivity is depending on the density and distribution of the brown to black color. As presented in Electrophoretic AssaysElectrophoretic Protein PatternC. tiglium nano-extract solely caused no alterations in the native protein pattern and no deviation detected from the control group. While AOM caused physiological alterations in the native protein pattern represented by hiding 3 normal protein bands with existence of the characteristic one that was identified in AOM induced colon cancer group at Rf 0.40 . Therefore, the lowest SI and highest GD values were recorded in AOM induced colon cancer group .As presented in C. tiglium nano-extract exhibited improvement in the protein pattern by hiding the characteristic bands with restoring 2 normal bands identified at Rfs 0.55 and 0.78  in nano-extract pre-treated group, one normal band identified at Rf 0.54  in nano-extract simult-treated group and 3 normal bands identified at Rfs 0.55, 0.63 and 0.78  in nano-extract post-treated group. Therefore, SI values increased with decreasing GD values in the nano-extract pre-treated, simult-treated and post-treated groups . Moreover, the relative quantities of the total protein bands declined significantly (P≤0.05) in AOM induced colon cancer group as compared to the control group (C. tiglium nano-extract increased the relative quantities significantly (P≤0.05) in all nano-extract treated groups with respect to AOM induced colon cancer group and restored quantities of the total bands to normal values. Treatment with ol group . TreatmeElectrophoretic Lipid Moiety of Native Protein PatternC. tiglium nano-extract alone caused no alterations in the native protein pattern and no deviation detected from the control group. While it was noticed that AOM caused physiological alterations in the lipid moieties of native protein bands represented by hiding the normal bands with existence of characteristic (unique) one identified at Rf 0.07 (B% 100.00 and Qty% 6.06) in AOM induced colon cancer group. Therefore, the lowest SI and highest GD values were recorded in AOM induced colon cancer group .As revealed in C. tiglium nano-extract exhibited improvement in the lipoprotein pattern by restoring one normal band identified at Rf 0.02 (B% 49.02 and Qty% 2.69) in the nano-extract pre-treated group with existence of one abnormal band at Rf 0.12 (B% 50.98 and Qty% 6.49). It restored 2 normal bands identified at Rfs 0.03 and 0.32  in the nano-extract simult-treated group with existence of one abnormal band at Rf 0.12 (B% 33.30 and Qty% 10.01). While in nano-extract post-treated group, nano-extract restored all the normal bands at Rfs 0.03 and 0.32  without existence of characteristic ones. Therefore, the SI values increased with decreasing the GD values in the nano-extract pre-treated, simult-treated and post-treated groups .Treatment with C. tiglium nano-extract increased the relative quantities significantly (P≤0.05) in all nano-extract treated groups with respect to AOM induced colon cancer group, relative quantities of the total bands restored to normal values in nano-extract post-treated group.As regard to the quantitative level, it was found that the relative quantities of the total lipoprotein bands declined significantly (P≤0.05) in AOM induced colon cancer group as compared to control group . AlthougElectrophoretic Calcium Moiety of Native Protein PatternC. tiglium nano-extract alone caused no alterations in the native protein pattern and no deviation detected from the control group. AOM caused physiological alterations in the calcium moieties of native protein bands represented by appearance of 2 characteristic (unique) bands identified at Rf 0.34 and 0.91  in AOM induced colon cancer group without hiding the normal ones. Therefore, the lowest SI and highest GD values were recorded in AOM induced colon cancer group .Data illustrated in C. tiglium nano-extract exhibited improvement in the calcium moiety of native protein pattern by hiding the abnormal characteristic bands in all nano-extract treated groups. Therefore, the SI values increased with decreasing the GD values in all the nano-extract treated groups .Treatment with C. tiglium nano-extract decreased the relative quantities significantly (P≤0.05) in all nano-extract treated groups with respect to AOM induced colon cancer group and restored quantities of the total bands to normal values. At the quantitative level, it was noticed that the relative quantities of the total bands increased significantly (P≤0.05) in AOM induced colon cancer group with respect to control group . TreatmeElectrophoretic isoenzymesElectrophoretic Catalase PatternC. tiglium nano-extract alone caused no abnormalities or deviation from control group. AOM caused physiological changes in CAT pattern represented by hiding the CAT1 type without appearance of characteristic bands. Therefore, the lowest SI and highest GD values were recorded in AOM induced colon cancer group . Treatment with C. tiglium nano-extract restored the physiological state of the electrophoretic CAT pattern through restoring the absent type (CAT1 type) in all nano-extract treated groups. Therefore, the SI values increased with decreasing the GD values in all the nano-extract treated groups .As illustrated in C. tiglium nano-extract increased the relative quantities significantly (P≤0.05) in all nano-extract treated groups with respect to AOM induced colon cancer group, quantities of the total CAT types restored to normal values in the nano-extract post-treated group. At the quantitative level, it was found that the relative quantities of the total CAT types decreased significantly (P≤0.05) in AOM induced colon cancer group as compared to control group . AlthougElectrophoretic Peroxidase PatternC. tiglium nano-extract alone caused no abnormalities or deviation from control group. As presented in C. tiglium nano-extract restored the physiological state of the electrophoretic POX pattern through restoring the absent type (POX3 type) in all nano-extract treated groups. Therefore, the SI values increased with decreasing the GD values in all the nano-extract treated groups .AOM caused physiological changes in the POX pattern represented by hiding POX3 type without appearance of characteristic ones. Therefore, the lowest SI and highest GD values were recorded in AOM induced colon cancer group . Treatment with C. tiglium nano-extract decreased the relative quantities significantly (P≤0.05) in all nano-extract treated groups with respect to AOM induced colon cancer group and restored quantities of the total POX types to normal values.As regard to the quantitative level, the relative quantities of the total POX types decreased significantly (P≤0.05) in AOM induced colon cancer group as compared to control group . C. tiglElectrophoretic Esterase PatternC. tiglium nano-extract alone caused no deviation from control group. While AOM caused physiological changes in the α-EST pattern represented by hiding α-EST2 type without appearance of characteristic ones. Therefore, the SI value (SI=50.00%) decreased with increasing GD value (GD=50.00%) in AOM induced colon cancer group. As shown in C. tiglium nano-extract restored the physiological state of the α-EST isoenzymes pattern through hiding the characteristic type with restoring the absent type (α-EST2 type) in all nano-extract treated groups. Therefore, the SI values increased with decreasing the GD values and became equal in all the nano-extract treated groups .Treatment with As regard to the quantitative level, it was noticed that AOM caused no significant changes in the relative quantities of the total α-EST types and values of the relative quantities of total α-EST types were approximately equal in all treated groups as compared to control group and AOM induced colon cancer group .C. tiglium nano-extract alone caused no deviation from control group. As presented in C. tiglium nano-extract restored the physiological state of β-EST isoenzymes pattern through hiding the characteristic type with restoring the absent type (β-EST1 type) in all nano-extract treated groups. Therefore, the SI values increased with decreasing the GD values and became equal in all the nano-extract treated groups .AOM caused abnormalities noticed physiologically in the β-EST pattern represented by hiding β-EST1 type with appearance of one characteristic  band identified at Rf 0.66 (B% 51.40 and Qty% 33.04). Therefore, AOM decreased the SI value with increasing the GD value  in AOM induced colon cancer group. The treatment with C. tiglium nano-extract increased the relative quantities significantly (P≤0.05) in all nano-extract treated groups with respect to AOM induced colon cancer group and restored quantities of the total β-EST types to normal values. At quantitative level, it was found that the relative quantities of the total β-EST types decreased significantly (P≤0.05) in AOM induced colon cancer group as compared to control group . TreatmeMolecular assayTP53 gene expression as compared to control group. In addition, levels of TP53 gene expression are significantly higher in C. tiglium nano-extract pre-treated and simult-treated groups when compared to that of control group . While the nano-extract post-treated group didn’t show any significant (P=0.417) change in level of TP53 gene expression than control group.As shown in APC gene expression as compared to control group. In addition, levels of APC gene expression are slightly significantly  higher in C. tiglium nano-extract pre-treated and simult-treated groups in comparison to control group. While the nano-extract post-treated groups didn’t show any significant (P=0.796) change in level of APC gene expression than control group.As illustrated in C. tiglium extract after incorporating Ag-NPs against colon cancer induced by AOM, it was found that AOM caused different deleterious effects at different hematological and biochemical levels during the induction process. It is necessary to use animal models for studying pathogenesis of colorectal tumors, as well as for evaluating the possible alternative treatments . Assessment of the haematological parameters can be used to determine the adverse effect on blood constituents of an animal and also it can be used to explain blood relating functions of chemical compounds/plant extract . Although the present study was designed to reveal efficiency of It is well known that AOM induced alterations in haematological status of rats and this attributed mainly to the oxidative stress that plays a role in haematological abnormalities and carcinogenesis progression . During the current study, it was found that level of RDW elevated significantly in AOM induced colon cancer. This was in agreement with Forhecz et al., (2009) who reported that the RDW level elevated in active malignancy that is accompanied by a prolonged inflammatory processes. In addition, the RDW value is closely related to colorectal cancer metastasis and it has been shown to predict advanced cancer stage . The MPV level is considered as an early marker of platelet activation. It elevated significantly in AOM-induced colon cancer. This was in agreement with Li et al., (2014) who reported that MPV was found to be independently associated with the presence of colon cancer and there was a positive correlation between MPV and stage of tumor-nodule-metastases. The PLT level elevated significantly in addition to WBCs and its differential count  in AOM induced colon cancer group. This might be attributed to the inflammatory reactions which stimulate the bone marrow to produce PLT and leukocytes especially lymphocytes because the leukocytes migrate to inflammation site and their attraction are facilitated by PLT . Moreover, neutrophils are considered as the first line of defense and rapidly recruited to inflamed loci in response to inflammatory mediators released at the injury site . Recent studies revealed that AOM can induce hepatotoxicity and develop liver injury , similarly to results of the current study. It was found that AOM caused elevation in levels of ALP, AST and ALP that are considered as the most common biomarkers used to evaluate liver functions. This was in accordance with Moharib et al., (2014) who reported that AOM caused liver damage occurred as a result of highly reactive electrophiles  that severely damage liver tissue causing necrosis and consequently leads to loss of membrane integrity. Therefore, cytoplasmic enzymes might leak from hepatocytes into blood at early stages of liver damage due to increasing membrane permeability.C. tiglium nano-extract restored these measurements to normalcy at all therapeutic modes (Protection and Treatment). This might be attributed to the presence of wide range of the polyphenolic compounds that increased after incorporating Ag-NPs as suggested by Abdelhady and Badr (2016) and supported by Aboulthana et al., (2019) who revealed that C. tiglium nano-extract contains higher concentrations of the active ingredients that exhibit antioxidant activity when compared to the crude one. Consequently, these compounds exhibited high ability to maintain integrity of cell membranes against reactive oxygen species (ROS) generated as a result of AOM injection.Silver It was found that AOM caused alterations in kidney functions. This was in agreement with Garba et al., (2007) who postulated that serum urea and creatinin were measured as indicators of kidney function and raising their concentrations are considered as an index of kidney dysfunction. Furthermore, it is well known that AOM is an active metabolite of the colon specific carcinogen 1,2-dimethylhydrazine (DMH) and it belongs to the hydrazine compounds which stimulate urea cycle and up-regulate biosynthesis of amino acids . Therefore, urea cycle might be expected to be up-regulated to remove excess ammonia derived from elevated amino acids. Also, amino groups (N-terminus) of amino acids that have been used as metabolic fuel are converted into urea through urea cycle resulting in increase of urea level . As regard to decline in the TP, significant increase in urea and creatinin levels act as an inhibitor of the synthesis of proteins . Moreover, levels of TP and albumin might be decreased significantly as a result of the liver injury that consequently leads to alterations in protein biosynthesis . In addition, urea and creatinin levels might be increased with lowering TP and albumin levels due to the severe inflammation occurred in kidney tissues as a result of AOM injection and this consequently leads to tubules degeneration and protein cast deposition within tubular lumina and tissue necrosis .C. tiglium nano-extract restored levels of the kidney functions to normal levels at all therapeutic modes. This might be attributed to presence of high concentrations of flavonoids that improve the renal physiology and possess diuretic and natriuretic properties, as well as exerting renoprotective effects that attributed to antiapoptotic and antifibrotic properties of these active constituents. In addition, levels of the TP and albumin restored to normal values due to the beneficial effect of nano-extract on liver tissues that became able to biosynthesize protein normally . Treatment with C. tiglium nano-extract decreased TC and T.Gs levels and this might be attributed to efficiency of the active constituents in the extract to develop lipolysis-stimulating agent in adipocytes . The lipolytic action of the extract was mainly mediated by phosphorylation of hormone-sensitive lipase (the key enzyme in the mobilization of fatty acids in adipocytes) and its activity is regulated post-transcriptionally by reversible phosphorylation by protein kinase A and this might aid in development of therapeutic strategy in preventing obesity .Lipids play a crucial role in maintaining the membrane integrity . In the present study, it was found that AOM caused significant elevation in levels of TC and T.Gs. This was in accordance with Yuvaraj et al., (2007) who emphasized that chemical agents that induce cancer development affect cellular lipids and cause abnormal lipid synthesis or defective degradation of lipids. Also, this might be attributed to deficiency of the lipoprotein lipase that helps in uptake of the T.Gs . In addition, Nalini et al., (2006) postulated that hypertriglyceridemia occurred as a result of tumors growth and it showed a stronger association with metastatic conditions. C. tiglium nano-extract decreased levels of the tumor markers and this might refer to the anti-tumor efficiency of the active constituents that increased after incorporating Ag-NPs in C. tiglium extract . These active constituents exhibited higher free radicals scavenging activity, reducing power and total antioxidant capacity that can inhibit the process of carcinogenesis effectively and prevent the development of invasive cancer. Therefore, they exhibit anti-tumor activity through inhibiting growth of cancer cells .It is well known that CEA and CA19-9 are the most common and specific markers of colon cancer. Levels of these markers are used not only for preoperative assessment of extent and outcome of cancer, but also postoperative monitoring of recurrence . Their levels are considered as the best serum marker for CRC and most thoroughly characterized tumor-associated antigens, in both biochemical and clinical aspects . In the present study, it was found AOM caused significant elevation in levels of these tumor markers. This was in accordance with many previous studies carried out by Mare et al., (2013) who postulated that AOM was categorized as a potent carcinogen and active intermediate of DMH and it leads to generation of ROS in the colon and hence, induce tissue lesions. Consequently, these ROS cause instability of colon cell metabolism leading to different changes in these tumor markers that are categorized as representatives of colon function. Moreover, elevation of serum CEA and CA 19-9 levels was significantly correlated with larger lesion size and multiplicity of adenomas . Treatment with C. tiglium nano-extract decreased levels of the inflammatory markers. This is in accordance with Jangid and Jadhav (2018) who documented that the anti-inflammatory effect of this plant extract against intestinal inflammation might be attributed to presence of the secondary metabolites that are well known by their ability to inhibit inflammatory reactions.The C-RP is called positive protein of the acute phase. Its level increases dramatically as a marker of tissue damage, inflammation and associated with prognosis in a variety of solid tumors . In the present study, the C-RP level elevated significantly in AOM induced colon cancer group. This might reflect ongoing inflammation and tissue damage more accurately than other biochemical measurements . Furthermore, Rajendiran et al., (2018) emphasized that elevating C-RP level might be attributed to the inflammatory effect of DSS that ingested via drinking water in combination with AOM during the induction process. It is known that MPO is an enzyme found predominantly in neutrophils and it is used as a good index of inflammatory response and tissue injury. Its activity is linearly related to neutrophil infiltration in colonic tissue . During the present study, it was found that the MPO activity is significantly higher in AOM injected group. This might refer to neutrophil infiltration that increased as a result of ingestion of DSS in association with AOM . Treatment with LDH belongs to the most important enzymes that play an effective role in the glycolytic pathway. It catalyzes conversion of pyruvic acid anaerobically and reversibly into lactic acid. It is used as an indicator for cancer development and also for many abnormalities in different tissues . During the current study, it was found that glucose level decreased significantly as well as, decline in LDH activity in the AOM induced colon cancer group. This was in agreement with Zhao et al., (1997) who postulated that glycolytic pathway increases in cancer tissue. This might be attributed to increasing number of cells that consume great glucose amount to get energy through glycolysis during cancer development and hence LDH activity consumed under anaerobic conditions . Moreover, growing cancer cells are able to destroy other tissues causing release of intracellular enzyme like LDH into blood stream by injury or death of cells and hence LDH decreased in cancer tissue . Also, calcium plays a primary and effective role in development of cell injury in vital organs at different pathological states. During the current study, calcium level increased in colon tissues of AOM induced colon cancer group. This was in accordance with Wulf (2000) who reported that calcium level increased as a result of the oxidative stress and disturbances of calcium homeostasis that might be induced in three different subcellular compartments including cytoplasm, mitochondria and the endoplasmic reticulum. Furthermore, marked elevation in level of cytoplasmic calcium was related to injury of colon tissues during the induction process. Nano-extract restored the glucose level, LDH activity and calcium level to normal values. This was attributed to efficiency of the active constituents to inhibit several metabolic intermediates and ROS formed during microsomal enzyme activation process . C. tiglium nano-extract. This might be attributed to nano-extract efficiency to prevent ROS generation in the colinic cells as presented by its ability to quench and scavenge DPPH and ABTs radicals’ formation . Dhanapal and Ping (2017) supported our findings and revealed that the antioxidant capacity of the plant extract increased after incorporating Ag-NPs. Due to its preference to adsorb antioxidant compounds onto surface of the Ag-NP, improving the scavenging ability.The MDA is considered as one of the final polyunsaturated fatty acids peroxidation products that are commonly known as marker of oxidative stress . AOM exhibited significant increase in the MDA concentration in colon tissues. This was in accordance with Khan and Sultana (2011) who reported that elevation of the colonic MDA exhibited mutagenic and genotoxic effect and may contribute to cancer development. Also, it induced protein damage, DNA fragmentation, gene mutations and loss of membrane integrity . Therefore, lipid and protein peroxidation products (LPO and TPC) increased significantly in AOM induced colon cancer group during the induction process of colon cancer. Antioxidant enzymes (CAT and GPx) provide the first line of cellular defense against the destructive effects of ROS. In the present study, it was noticed that AOM caused significant decline in total antioxidant capacity and activities of these antioxidant enzymes. This was in accordance with Waly et al., (2016) who emphasized that AOM has the ability to initiate oxidative stress through producing extremely ROS and by modifying activities of the antioxidant enzymes. Furthermore, AOM induced the oxidative stress through depletion of intracellular reduced glutathione . The plant extract contains wide range of the active compounds which exhibit antioxidant properties in a dose dependent manner . Moreover, due to the phenolic compounds available in this extract leading to antioxidant activity, it can scavenge of free radicals such as hydroxyl radical which is the major cause of peroxidation reactions . The colonic antioxidant profile was restored to normal levels by Conventionally, carcinogenesis is well defined by three stages: initiation, promotion and progression . In the present study, the H and E staining of colonic tissues was carried out for histopathological examination and it was found that severe histological abnormalities were noticed in colonic mucosa of the AOM-treated rats. This was in accordance with Saleem et al., (2015) who emphasized that oxidative stress and depletion of the antioxidant enzymes were responsible for inflammatory cells infiltration and severe histopathological alterations noticed in AOM induced colon cancer group. Moreover, AOM is a metabolite of DMH, whose mechanism of preneoplastic lesions induction is caused by the increased expression of c-fos gene and reduced c-myc gene expression, as well as alterations in the mutated K-ras gene expression similar to those observed in spontaneous carcinogenesis in humans . Also, alterations in architecture of colonic mucosa with typical inflammatory changes such as crypt and surface epithelial loss as well as infiltration of inflammatory cells and complete destruction of the epithelial architecture might refer to the effect of DSS that administrated via drinking water in association with AOM during the induction process . C. tiglium nano-extract minimized severity of the inflammatory responses that is considered as criteria of malignancy and stage of colon cancer in nano-extract simult-treated group while these inflammatory responses were prevented completely in the post-treated group. Due to presence of phenolic compounds that are considered as part of a complex defense mechanism against wide range of stressors, they accumulate in response to these deleterious factors . These phenolic compounds are well known as good antioxidant agents due to their capability of singlet oxygen-quenching and radical scavenging activity and incorporation of Ag-NPs into the extract increased the antioxidant efficiency . Herein, treatment with nano-extract protected the colonic cells from damage and dysfunction that induced by oxidative stress. This might refer to the beneficial efficiency of C. tiglium nano-extract to protect cells by maintaining membrane permeability through preventing glutathione depletion and hence inhibiting peroxidation reactions.In addition, cytokeratin 20 was expressed severely in colonic tissues of AOM induced colon cancer. This was in accordance with Derry et al., (2014) who showed that cytokeratin 20 is considered as an additional colon biomarker and expressed virtually in epithelial cells in all cases of colorectal carcinomas. The present study revealed that Electrophoresis is standard technique for separating, identifying and quantifying of different proteins and it is considered as the most commonly tool used to analyze stoichiometry of a specific subunit of a protein complex . Mutations may occur at qualitative level by disappearance of normal bands and appearance of one or more abnormal ones. Otherwise, the alterations may occur quantitatively by remaining normal bands but with altering their quantities. The SI is only correlated to the qualitative alterations . The SI is inversely proportional to the genetic variation. The low SI values between control and all the treated groups indicate differences in bands number and arrangement .C. tiglium nano-extract showed ameliorative effect against the alterations induced by free radicals’ attack in different native electrophoretic protein patterns. This might refer to presence of the active polyphenolic constituents that increased as a result of incorporating Ag-NPs as reported by Aboulthana et al., (2019).Proteins represent the key players inside the cells. They are susceptible to be oxidized and this depends on relative content of oxidation-sensitive amino acid residues . In the present study, AOM caused alterations in native protein pattern. This was in agreement with Yasui and Tanaka (2009) who suggested that the alterations in native protein might be attributed to interaction of AOM with proteins. Furthermore, Aguilar-Mahecha et al. (2002) reported that the electrophoretic alterations might refer to deleterious effect on RNA transcripts, altering their amounts, localization or translation. Also, the modifications in overall native protein pattern might be related to altering DNA organization and hence changing DNA function and hence protein expression . Lipids are the most sensitive part of the cellular macromolecules . They are the major components of chromosomes, chromatin and nuclear matrix . During the present study, AOM altered the native lipoprotein pattern severely. This might refer to depletion of the antioxidant defenses and accumulation products of the oxidative stress that attack the lipid portion through inducing formation of the ROS as suggested by El-Sayed et al., (2018). Consequently, this leads to oxidative modifications and denaturation of the lipid moieties of proteins . Calcium-binding proteins categorized as acidic proteins with low molecular weights. They exhibited inhibitory effect hydroxyapatite formation leading to alterations in the mineralization process . Furthermore, they exhibit an effective role in resistance of the tissues against the toxic agents . The alterations in these proteins might occur as a result of abnormal tissue mineralization . During the current study, it was noticed that AOM caused changes in calcium moieties of the native protein. This was in agreement with El-Sayed et al., (2018) who postulated that these qualitative and quantitative abnormalities in calcium moieties of the native protein might be attributed to the oxidative stress and generation of ROS that convert an active hydrogen atom from these biologically active macromolecules. Furthermore, these alterations might be related to the significant increase in calcium concentration in the colon tissue as described during the current experiment and this leads to stimulating the oxidative stress and hence leads to variations in this protein pattern. C. tiglium nano-extract showed antioxidant efficiency against the alterations in electrophoretic CAT and POX patterns. This might be due to enhancing the polyphenolic compounds that exhibit scavenging activity against ROS attack .Antioxidant enzymes are considered as tissue dependent tools of the antioxidant system. Their activities vary from tissue to tissue. The changes in antioxidant system might refer to attack of the ROS targeting protein contents and altering metabolic pathways . In the current study, AOM caused abnormalities in the electrophoretic antioxidant isoenzymes. This was in agreement with Aboulthana et al., (2016) who suggested that changing the fractional activity of different isoenzymes seemed to be correlated with the changes in rate of protein expression secondary to DNA damage induced by ROS. Consequently, this leads to structural changes in protein portion of native enzymes . No changes occurred in the enzymatic activities, if there were no alterations in the protein expression . Furthermore, these alterations might be related to the significant decrease in the glucose level and LDH activity in the colon tissue as described during the current experiment and this referred to altering the glycolytic pathway and hence changing the glycation process that consequently inhibits enzyme activity . Moreover, AOM altered the electrophoretic CAT pattern. This might be attributed to binding of this toxic substance to native macromolecules inducing secondary structural changes in the enzyme . Also, the alterations in the electrophoretic POX pattern might refer to the oxidative stress and the peroxidation reactions . Esterase-like albumin activity used as prognostic marker for various choronic diseases . It is expressed as several molecular forms that can be distinguished by their molecular weights and hydrodynamic properties . It can be visualized by staining the substrate using α- and β-naphthyl acetate in the presence of Fast Blue RR salt as a dye coupler . The alterations in electrophoretic EST pattern might be due to enhancement of oxidative stress markers which consequently results in altering albumin structure and function . Also, the characteristic changes in EST pattern might refer to changing glycosylation of different ESTs in bile duct ligation liver. This consequently reflects changes in the glycosylated EST forms. The molecular EST pattern that remains unaffected, displays normal glycosylation pattern . The glycosylation process that occurred abnormally increases probability of protein degradation and affected EST stability . Due to increasing concentration of the total polyphenols after incorporating Ag-NPs, nano-extract exhibit antioxidative properties that could play an important role in maintaining integrity of macromolecules against oxidative reactions . In addition, these polyphenols stimulate activity of antioxidant enzymes to overcome attack of free radicals targeting these biomacromolecules .TP53 and APC are the most sensitive genes susceptible to harbor mutations induced colorectal cancers involving the distal colon . In the current study, it was found that AOM induced colon cancer caused significant elevation in relative expression of TP53 gene and this was in agreement with Nambiar et al., (2002) who reported that level of TP53 gene expression might be attributed to increase accumulation of TP53 protein within the tumor cells. Moreover, it was suggested that elevation of the TP53 gene expression might refer to stability of TP53 protein that increased as a result of the aberrant alterations in TP53 acetylation and/or phosphorylation status and this suppresses its binding to DNA consensus sequences. Also, transactivation and repression function of TP53 protein might be inhibited by alternative mechanisms. Suppression of TP53 activity in AOM-induced colon cancer may refer to differences in its post-translational modification . This was supported by Nambiar et al., (2002) who illustrated that TP53 protein isolated from AOM-induced colon tumors was found to migrate more slowly on gel relative to the active form of TP53 isolated from normal cells. APC exhibits its normal functions by promoting cellular differentiation of intestinal lineages including mucus-producing goblet cells . It is considered as one of the tumor suppressor genes which are susceptible to various mutations . It is the most frequent mutated gene expressed as a result of precancerous lesions in colon carcinogenesis . Cancer progression is usually associated with irregularities in cell cycle . Recent study directed to search in nature to find evidence supported experimentally suppression of the concomitant involvement of cell cycle by mean of apoptosis to phytochemicals with cell-cycle modulatory effects . The APC gene and this was supported by the results obtained by Grivennikov et al., (2012) who reported that level of APC gene expression was noticed significantly greater in AOM induced colon cancer group. This attributed to role of APC that acts as a central gatekeeper protein in colorectal tumorigenesis for regulating multiple cancer pathways. The alterations in expression of APC gene lead to changes in expression of APC protein and abnormalities in cell cycle and hence promoting colon cancer .During the current study, it was noticed that AOM caused significant elevation in relative expression of C. tiglium nano-extract decreased level of the relative expression of TP53 and APC genes in the post-treated group to normal level. Although it could not decrease their levels in the pre-treated and simult-treated groups to normalcy, it decreased their levels significantly as compared to AOM-induced cancer. This was in accordance with the results obtained by Li et al., (2016) who verified that extract induced cellular apoptosis through regulating the apoptotic indexes, apoptotic rates and apoptosis-associated gene expressions. The extract is rich in the croton alkaloid and diterpenoids that have been found to inhibit cell proliferation or induce programmed cell death to control the growth of malignant cells . Concentrations of these phyto-constituents increased after incorporating Ag-NPs . Consequently, the cytotoxic efficiency of nano-extract against growth of human colon cancer cells increased in comparison with the crude ones. Therefore, efficiency of C. tiglium extract was evaluated as a new plant-derived anticancer agent after incorporating silver nanoparticles during the current study.Treatment with C. tiglium nano-extract exhibited ameliorative effect against AOM induced colon cancer by decreasing levels of the hematological and biochemical measurements and restored their levels to normal values in all nano-extract treated groups. It restored levels of the tumor (CEA and CA 19-9) and inflammatory (C-RP and MPO) markers to normalcy in post-treated group. Moreover, it improved the antioxidant system by increasing activities of the antioxidant enzymes (CAT and GPx) with decreasing products of the peroxidation reactions (LPO and TPC) in all nano-extract treated groups and restored their levels to normal values in post-treated group. It minimized severity of the inflammatory reactions in all nano-extract treated groups and prevented it completely in nano-extract post-treated group. Moreover, it prevented the qualitative alterations in the different native patterns and isoenzymes in nano-extract post-treated group through hiding abnormal bands with restoring normal ones. Also, it prevented the quantitative alterations in all nano-extract treated groups and restoring the relative quantities of the total native bands to control values. As regard to the molecular assay, nano-extract decreased expression of the tumor suppressor genes (TP53 and APC) in all nano-extract treated groups but it restored their expression levels to normal values in nano-extract post-treated group.In conclusion, the study concluded that


With the advent of highly sensitive technologies such as tandem mass spectrometry and next-generation sequencing, recombinant antibodies are now routinely analyzed for the presence of low-level sequence variants including amino acid misincorporations. During mAb cell culture process development, we found that proline was replaced with the non-canonical amino acid, hydroxyproline, in the protein sequence. We investigated the relationship between proline content in the cell culture media and proline sequence variants and found that the proline concentration was inversely correlated with the amount of sequence variants detected in the protein sequence. Hydroxyproline incorporation has been previously reported in recombinant proteins produced in mammalian expression systems as a post-translational modification. Given the dependency on proline levels, the mechanism was then investigated. To address the possibility of co-translational misincorporation of hydroxyproline, we used tandem mass spectrometry to measure incorporation of stable-isotope labelled hydroxyproline added to the feed of a production bioreactor. We discovered co-translational misincorporation of labelled hydroxyproline in the recombinant antibody. These findings are significant, since they underscore the need to track non-canonical amino acid incorporation as a co-translational event in CHO cells. Understanding the mechanism of hydroxyproline incorporation is crucial in developing an appropriate control strategy during biologics production. Chinese hamster ovary (CHO) cells are the main mammalian expression system for protein production used by the biopharmaceutical industry , 2. A prProtein translation in mammalian cells is typically a high-fidelity process. This process relies on accuracy in the esterification of a cognate amino acid to tRNA upon recognition by the appropriate aminoacyl-tRNA synthetase. In addition, multiple pre- and post-transfer proofreading steps can result in hydrolysis of a non-cognate amino acid, thus reducing the probability of translation errors , 11. AmiE. coli prolyl-tRNA synthetase can misincorporate hydroxyproline albeit at much lower rates than proline [In addition to proteinogenic amino acids, non-proteinogenic amino acids and rare PTMs can also be detected in the protein sequence –21. One  proline . To the Our work describes the characterization of a subset of proline misincorporations substituted with hydroxyproline, which is found in a recombinant antibody produced in CHO cells using a fed-batch production process. We found hydroxyproline misincorporation in a proline concentration-dependent manner and demonstrated that the mechanism included co-translational misincorporation by using deuterium labelled hydroxyproline. Although misincorporation of non-proteinogenic amino acids is not widely monitored in biotherapeutics, this work suggests that non-proteinogenic amino acids can be misincorporated by mammalian expression systems and the knowledge of the mechanism will be critical to develop effective mitigation strategies.2 incubator at 37°C at 125rpm shaking speed. Bioreactor production was performed using small-scale (Applikon Inc.) and ambr250 (Sartorius AG) bioreactors. A fed-batch production process was carried out by inoculating a production bioreactor with a fixed split ratio from a targeted N-1 culture of appropriate volume. Cells were inoculated in the basal medium and a proprietary feed was added to the bioreactor based on a feeding schedule. Process parameters such as pH, dissolved oxygen, agitation and temperature were controlled. The growth of cells was monitored via viable cell density (VCD) and viability measurements everyday using a Vi-CELL XR automated cell counter (Beckman Coulter). Cell culture supernatants were collected, and the samples were analyzed for titer and metabolite concentrations using a metabolite analyzer (Roche AG). Production was typically carried for 14 days, and final day cell culture samples were purified and submitted for sequence variant analysis.Industrially relevant CHO cell lines producing recombinant monoclonal antibody (mAb) were used for all the experiments performed in this study. Cells were cultured in a proprietary basal medium, and scaled up in shake flasks (Corning Inc.) maintained in a 5% COThe harvest cell culture fluid samples (HCCF) were purified by affinity chromatography using immobilized Protein A resin  according to the manufacturer’s recommended procedure. The mAbs were eluted under acidic conditions with sodium acetate buffer at pH 3.4. The mAb concentration was detected by UV absorption at 280 nm. Protein A purified mAb was used for sequence variant analysis.Deuterium  labelled trans-4-hydroxy-l-proline (Cambridge Isotope Laboratories Inc) at 96% purity was added to the bioreactor feed media after the unlabelled free hydroxyproline was removed from the chemically defined portion of the feed formulation. The concentration of deuterium labelled hydroxyproline was reduced to 85% of the concentration of free hydroxyproline in the original bioreactor feed media. For side-by-side comparisons, the hydroxyproline concentration in the control conditions were adjusted accordingly. This adjustment was performed to ensure that sufficient volume of labelled hydroxyproline feed was available across the duration of the experiment based on limited quantities of the D5 labelled hydroxyproline for two replicate bioreactors. For the labelled hydroxyproline experiment, bioreactors were harvested early on day 8 for misincorporation analysis, as day 8 samples previously demonstrated measurable proline substitutions.HCCF samples were analyzed for free amino acids using spent media analysis. Amino acids were derivatized using 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AMC), and separated by reversed-phase chromatography using Waters Acquity UPLC BEH C18 column (Waters Corp.) with UV absorbance detection at 260 nm. Individual amino acids in each sample were quantified against each respective standard curve.HCCF samples were diluted using an appropirate diluent and a known concentration of isotopic amino acid standard was added. Separation was performed by capillary electrophoresis using the ZipChip system (908 Devices Inc) with detection by Q Exactive HF-X mass spectrometry. Amino acid concentrations were quantified using the isotopic standard as a single point calibrant.Protein A purified mAb samples were reduced and alkylated with dithiothreitol (DTT) and iodoacetic acid (IAA), respectively, under denaturing conditions with guanidine hydrochloride followed by digestion using the endoproteinase trypsin. The resulting peptide mixture was separated on a C18 reversed phase column using a gradient of increasing organic concentration in a water/acetonitrile solvent system. Peptides were detected by UV and online mass spectrometry (MS) with a Thermo Q-Exactive mass spectrometer. Peptides were detected in MS survey scans, quantified by extracted ion chromatogram (XIC) integration and validated by MS/MS scans using Thermo Xcalibur software. Sequence variants in these peptides were evaluated in a similar way and the amino acid substitutions were localized using MS/MS scans. The XIC ratio of a sequence variant was calculated as the ratio of the XIC peak area for the variant peptide relative to the total XIC peak area for the variant and nominal peptide. This value per residue was added across the analyzed protein sequence to calculate a summed sequence variant/misincorporation percentage for each substituted amino acid.Amino acid substitutions, primarily at proline residues, were observed in recombinant antibodies produced from four CHO cell lines  in standard fed-batch production during early-stage development. In order to understand the mechanism of proline substitutions, cell culture performance of cell line D was analyzed further.  show theTo investigate co-translational misincorporation of hydroxyproline, we replaced hydroxyproline in the cell culture feed medium of cell line D with deuterium labelled hydroxyproline. If the co-translational hypothesis is correct, then we would expect that when proline levels fall below a certain level in cell culture, proline tRNAs will be misacylated with hydroxyproline and hydroxyproline will be misincorporated into the primary sequence of the recombinant protein. The presence of the labelled hydroxyproline can then be verified and quantitated with standard LC-MS/MS methodologies. Due to the labelled hydroxyproline availability, 85% of the original hydroxyproline concentration in the feed was chosen, and a control condition with the same concentration of unlabelled hydroxyproline was included. In addition, a control condition with 100% unlabelled hydroxyproline added to the feed formulation was run to monitor process consistency and ensure comparable performance in this experiment with respect to prior runs. The bioreactors incorporating the isotope-modified feed followed the same feed schedule used previously but with early termination on day 10, and day 8 HCCF samples were used for sequence variant analysis. Cell growth and antibody production in the controls and labelled hydroxyproline bioreactors were consistent as shown in  demonstrSequence variant analysis confirmed proline misincorporation to both alanine and hydroxyproline in all the bioreactors. Misincorporation rates in the 100% control sample were comparable to previous experiments at similar bioreactor scale (11.2% vs 8.5%) and summed proline substitutions was approximately 10% for the 85% hydroxyproline bioreactors. Both unlabelled and labelled hydroxyproline were detected in the HCCF sample from the labelled hydroxyproline fed bioreactor by performing spent media analysis, confirming the misincorporation of hydroxyproline into the recombinant antibody sequence. As expected, based on the purity of the labelled D5-hydroxyproline (96%), a prominent M-1 peak was detected in the MS1 precursor scan which corresponds to the D4-hydroxyproline putative impurity. A binomial model was used to account for under-labelled hydroxyproline and the total hydroxyproline misincorporation level was calculated using the sum of D4- and D5-hydroxyproline intensity . MS/MS sIn this study, we identified proline sequence variants substituted with hydroxyproline in recombinant antibodies produced in CHO cells and investigated the mechanism of misincorporation. Sequence variants mainly arise from DNA mutations, post-translational modifications or mistranslation due to tRNA misacylation or misdecoding (codon mispairing or wobble) . MisincoE. coli [E. coli [Sequence variants caused by misincorporation of non-canonical amino acids like norvaline and norleucine have been previously reported in recombinant proteins produced in E. coli . In addi[E. coli . Non-pro[E. coli –33. Reco[E. coli –36.E. coli can be driven by the lack of cognate amino acid as well as factors that promote synthesis of the non-cognate amino acids in the culture medium [Non-proteinogenic amino acid misincorporation requires sufficient concentration of the substituting amino acid in comparison with the cognate amino acid, as well as the propensity for mischarging by amino acyl-tRNA synthetase. Misincorporation of non-proteinogenic amino acids in recombinant proteins produced in e medium –40. Spene medium , 42. Adde medium –45.Given the presence of hydroxyproline and prior evidence of prolyl tRNA mischarging proline analogs, we tested the hypothesis that the hydroxyproline substitutions were co-translational misincorporations. Our data showed that hydroxyproline can be incorporated into CHO expressed antibody, most likely via co-translational misincorporation. Unlabelled hydroxyproline was also found in the recombinant protein sequence consistent with the presence of unlabelled hydroxyproline in cell culture media and feed. The fraction of labelled hydroxyproline relative to total hydroxyproline in HCCF was measured using spent media analysis, at an average of 59 percent from days 6 to 8. Analysis of multiple peptides showed that the fraction of labelled hydroxyproline relative to total hydroxyproline was at 65.7–72.9 percent suggesting that a significant fraction of unlabelled hydroxyproline was present in the protein sequence similar to the percentage measured in the HCCF using spent media analysis. The low level of misincorporations distributed randomly across the protein sequence, dependence on proline levels and the incorporation of labelled hydroxyproline suggests co-translational misincorporation as a mechanism for hydroxyproline incorporation into recombinant proteins produced in CHO cells.This study has conclusively shown that CHO cells, the most widely used expression host for recombinant therapeutic expression, can incorporate non-proteinogenic amino acid hydroxyproline into proteins using an additional mechanism, which is mistranslation based misincorporation. This finding is significant since non-proteinogenic amino acid misincorporation is not routinely monitored during analytical characterization, and cell culture components can potentially provide a source for some of these amino acids, like hydroxyproline. This route of misincorporation into recombinant proteins necessitates a different approach to the development of mitigation and control strategies, for example, controlling or eliminating the levels of non-cognate amino acids available in the cell culture media. Mitigating co-translational misincorporation of non-proteinogenic amino acids will require development of sensitive detection assays to confirm the mechanism and analyze the relationship between amino acid concentrations and misincorporation levels. Understanding the risks associated with the incidence and impact of these types of misincorporation will be necessary to develop a sound control strategy for controlling misincorporation in recombinant protein-based therapeutics.S1 FigAn MS2 spectra corresponding to each of the peaks from (TIF)Click here for additional data file.S2 FigMS2 spectra corresponding to the D0 and D5 species taken at the same retention time are shown with b and y-ion annotation. The offset caused by the labeling is visible in the b2 ion.(TIF)Click here for additional data file.


Polyaniline (PANI) layers are electrochemically obtained in the presence of four polysulfonic acids with different rigidities of the polymer backbone-iso-(and tere-)poly--diphenylene-iso(tere)-phthalamide (i-PASA and t-PASA), polystyrenesulfonic acid (PSSA) and poly(2-acrylamido-2-methyl-1-propanesulfonic acid) (PAMPSA). Combined microgravimetric (EQCM) and electrochemical measurements are carried out in the course of polymerization and repetitive redox switching. It is found that after synthesis PASA-doped PANIs shows good stability with low exchange of mass in the course of voltammetric scans, while PAMPSA-doped PANI contains a large amount of water that gradually becomes expelled in the repetitive redox switching. PANI obtained in the presence of PSSA takes an intermediate position with respect to mass exchanged in the electrochemical redox process. XPS studies are used to obtain data for the extent of doping of the different PANI materials. The results show high doping level (about 0.5) for PASA- and PAMPSA- and lower level (0.32) for PSSA-doped PANI layers. Repeated electrochemical studies carried out with the specimens investigated by XPS after long-term storage in the dry state give evidence for structural rearrangement, perfect recuperation of the initial electrochemical activity and high stability of the electrochemical response. Polyaniline (PANI) is one of the most studied conducting polymer materials that provokes continuous interest due to multiple emerging applications ,2,3. PANThe aim of the present study is to compare PANI layers obtained in the presence of four different polysulfonic acids with respect to kinetics of electrochemical polymerization, electrochemical redox behavior (including type of ionic and solvent fluxes) and extent of doping. To achieve this aim electrochemical, microgravimetric and XPS studies are carried out on the same samples in order to obtain self-consistent information. Furthermore, data for the electrochemical activity of the four types of PANI samples are obtained both directly after synthesis and after XPS studies and long-term storage in the dry state.XPS studies are commonly used to obtain the extent of doping of PANI materials whereas the electrochemical redox behavior of PANI-coated electrodes is used to evaluate the so-called redox number. The latter is based on data for the polymerization and redox charges and should be also an estimate for doping. PSSA-doped PANIs were studied by means of XPS in several cases ,14,15,16Mw = 2,000,000, 15% aqueous solution) and sulfuric acid  were used for preparing the synthesis and characterization solutions. PSSNa  was converted into H+-form using ion-exchange column. Both PASA polyacids were obtained from laboratory synthesized i-PASA and t-PASA sodium salts by converting them into the H+-forms according to the procedure described in [Mw = 474.32 g/M for t-PASA and i-PASA, Mw = 184.14 g/M for PSSA (flexible), and Mw = 207.24 g/M for PAMPSA. The polyacids were diluted with distilled water to obtain 0.05 M PAMPSA or PSSA, 0.025 M i-PASA or t-PASA aqueous solutions. The concentration of i-PASA and t-PASA was reduced twice in order to keep the same aniline per sulfonic group ratio in all solutions. Freshly distilled aniline (0.025 M) was added in the polyacid solutions and they were let for several hours  on a magnetic stirrer.Aniline , PAMPSA , ribed in . The molQp. PANI specimens with Qp = 5 and Qp = 25 mC were obtained in each polymerization solution. After polymerization the redox behavior of the PANI coatings was investigated in 0.5 M H2SO4 by cyclic voltammetry at a scan rate of 50 mV/s. These measurements were repeated on the same specimens after XPS measurements and log-term (12 months) storage in the dry state.Polyaniline was electrodeposited at constant polymerization potential (E = 0.7 vs. Ag/AgCl) in the presence of the different doping ions by interrupting the polymerization at fixed values of the anodic charge, 2 and sensitivity of 4.43 μg/kHz were used as working electrodes for potentiostatic polymerization and voltammetric redox experiments. An Ag/AgCl electrode was used as reference and a gold wire as counter electrode.Simultaneous microgravimetric and electrochemical measurements were carried out by means of Metrohm-Autolab BV  EQCM device coupled to Autolab 302N potentiostat/galvanostat. Gold-coated quartz resonators (6 MHz) with surface area of 0.361 cm−7 Pa. The spectra were recorded using achromatic Al Kα radiation with a photon energy of 1486.6 eV and a charge neutralization system. The energy scale was calibrated by normalizing the C 1 s line of adsorbed adventitious hydrocarbons to 284.6 eV. The binding energies (BE) were determined with an accuracy of ±0.1 eV. The commercial data-processing software of Kratos Analytical Ltd.  was used to calculate the concentrations of the different chemical elements (in atomic %) by normalizing the areas of the photoelectron peaks to their relative sensitivity factors. The deconvolution of the high-resolution element spectra was performed by the ESCApe™ software .X-ray photoelectron spectroscopy (XPS) was carried out by means of an AXIS Supra electron spectrometer  with base vacuum in the analysis chamber of ~10Current transients obtained in the course of electrochemical polymerization of aniline in the four polymerization solutions and corresponding results from the microgravimetric measurements are presented in The type of dopant affects markedly the polymerization kinetics—very fast growth is observed for PAMPSA- and t-PASA-doped PANI whereas i-PASA and PSSA slow down the polymerization. The microgravimetric data show a similar frequency shift with polymerization charge for i-PASA- and PSSA- doped layers whereas t-PASA and especially PAMPSA-doped layers show significant Δf decrease. The most striking effect is observed when comparing growth in the presence of i-PASA and t-PASA which have the same molecular weights and nevertheless result in markedly different frequency response throughout polymerization. Furthermore, at long times PAMPSA-doped PANI shows the largest frequency shift although i-PASA and t-PASA have higher molecular weight per repeat unit than PAMPSA. Apparently, the experimentally obtained frequency shifts do not follow the molecular weights of the doping ions (PSSA<PAMPSA<i-PASA = t-PASA) and thus additional effects such as incorporation of solvent molecules, non-stoichiometric insertion of doping ions (overdoping) and/or dopant-dependent polymerization efficiency should be taken into consideration. Mapp = zFΔm/Qp where Δm is the mass change at passed polymerization charge Qp, F is Faraday’s constant and z is the number of electrons involved in the elementary electrochemical electrodeposition step. The oxidation of the aniline monomer takes two electrons per oxidized unit and additionally there is partial oxidation of the nitrogen atoms in the PANI backbone with maximal value of the number of electrons per monomer equal to 0.5. app calculated based on Sauerbrey’s equation for the initial parts of the Δf vs. Q dependencies (for Q between 0 and 1 mC) by taking the maximal possible extent of oxidation . The data for t-PASA are evaluated also at a later polymerization stage (for Qp between 10 and 11 mC) because of the marked decrease of the slope dΔf/dQp observed for this dopant. The last column in For thin PANI layers see  a linearMapp values observed in the PAMPSA and t-PASA cases, overdoping of polysulfonate species and/or significant solvent incorporation should be assumed. PSSA- and t-PASA(advanced stage)-doped PANIs take an intermediate position with about 25% to 33% overweight with respect to the theoretical expectation. The analysis of the microgravimetric data so far performed indicates that the most flexible dopant (PAMPSA) and the rigid t-PASA  show the largest initial overstoichiometric mass gain, which is persistent for PAMPSA and fades out for t-PASA. For PAMPSA this seems to be an expected effect due to its flexibility and hydrophilicity. The effect of t-PASA should be a different one. Due to its rigidity t-PASA cannot easily adjust to the likewise rigid PANI strands and thus accommodation of both structures seems to take place at a later stage of the building of the bulky net-like polymer structure in which PANI fragments serve as cross-linking chains for several polyacid macromolecules [The comparison of the data for the apparent molar mass obtained experimentally (column 5) and calculated at stoichiometric insertion of doping ions (column 6) shows close values for the case of i-PASA and thus no over stoichiometric gain of mass is found. In contrast, based on the large experimental olecules ,23. It wolecules  that t-PThe electrochemical redox behavior and stability upon several switching between reduced  and semi-oxidized  states was studied by means of cyclic voltammetry in sulfuric acid solution. The voltammetric measurements for thin and thick PANI layers obtained in the presence of the four polyacids are shown in y characterizing the oxidation state of PANI. By equating the number of moles of aniline taking part in the polymerization reaction and in the redox process the following relationship is easily obtained y = 2Qox/(Qp− Qox), where Qox is the oxidation charge under the voltammetric peak obtained in the course of redox cycling. This relationship holds provided 100% polymerization efficiency. The data calculated for thin and thick PANI layers are presented in Qox is calculated in the potential interval −0.14 to 0.4 V for all specimens.) Thick PANI layers show in all cases higher y values very probably due to the possibility to obtain longer chains and to achieve advantageous structural re-arrangement with the advancement of growth. Larger y values are obtained for t-PASA- and i-PASA- followed by PAMPSA-doped PANIs whereas rather low y values characterize both thin and thick PSSA-doped layers. These results correlate with the observed sharp oxidation peaks and the presumed more ordered structure of PANIs obtained with PASA-dopants. In addition, it should be mentioned that, compared to PAMPSA, PASAs-doped PANI films exhibit much more intensive changes of absorbance in the NIR-range during variation of the potential within the range of leucoemeraldine to emeraldine transition [The oxidation charge in the voltammetric curves is used to calculate the so-called redox number ansition ,25. app = 15–16 g per mol charged species for i-PASA and t-PASA, 28 g/mol for PSSA and 49 g/mol for PAMPSA. These values correspond roughly to one (for PASAs), one and half (for PSSA) and three molecules (for PAMPSA) of water involved in the accommodation of one charged species in the PANI backbone. It is apparent that for both t- and i-PASA-doped PANI there is almost no structural relaxation of the polymer structure upon redox switching.Microgravimetric results obtained in the course of single voltammetric cycles measured at the four types of thick PANI layers are presented in The stability of the polyacid-doped PANIs upon repetitive redox switching was checked by measuring several voltammetric scans a–d. ThesFurther electrochemical studies during redox switching  in somew+) (peak c) and protonated imine ( = NH+) (peak d) species with peak positions found at 398.5 eV, 399.7 eV, 401.1 eV and 402.2 eV, respectively. The doping level of PANI is usually estimated by the ratio of the charged nitrogen species to the total nitrogen content. In our case, however, the doping species (PASA and PAMPSA) contain also nitrogen (with stoichiometry S:N = 1:1) which should be excluded from the calculation for the doping level. Thus, for the PASA and PAMPSA cases the total nitrogen atomic content was reduced by extracting the equivalent of the available S content. Therefore except for PSSA the ratio N(c+d)/(N–S) was used to estimate the doping level. The doping level for PSSA-doped PANI (N(c+d)/N) amounts to 0.32 and is in the range of values (0.30–0.47) obtained for this system in former studies (see tot/(NtotStot) (for PAMPSA and PASAs) and Stot/Ntot for PSSA (column (11) in XPS studies were completed on the PANI specimens used already for coupled electrochemical and microgravimetric measurements. The elemental composition and N 1s deconvoluted spectra  were obtdies see . The datdies see  which medies see . Further+ and NH = + oxidation processes which means that the peak due to NH+ formation cannot be clearly resolved from the charge of further oxidation. This point remains however open and requires further combined electrochemical and XPS studies.Finally, one of the specific opportunities in our combined electrochemical and XPS study is the possibility to correlate the doping (based on the amount of charged N species from deconvoluted XPS spectra) and the extent of electrochemical oxidation in the leucomeraldine to emeraldine oxidation transition. The electrochemically obtained redox numbers, y  are systThe comparative study carried out in the present work shows marked differences in the polymerization behavior of PANI in the presence of two polysulfonic acids with identical chemical composition and different rigidity of the polymer backbone. The rigid t-PASA seems to act as a perfect template to initiate polymerization of aniline without constraints. The net-like structure of the interpolymer complex seems to open enough place for access to the oxidizing aniline units. The process slows down at a later stage and so does the observed overstoichiometric insertion of mass. In comparison, i-PASA decelerates the polymerization from the very beginning due probably to steric hindrances of the more flexible dopant chains. Despite this difference both PASA-doped PANIs show perfect stability upon electrochemical redox switching and very low exchange of mass (presumably one water molecule per PANI oxidative charge). Data obtained from XPS experiment indicate to about 50% doping level of the PASA-doped PANIs.PAMPSA-doped PANI is characterized by fast polymerization accompanied by massive ingress of solvent molecules that become later gradually expelled in the course of electrochemical redox switching. Apparently, the fast kinetics of PANI growth and random-coil conformation of PAMPSA ,35 does y values. The combined electrochemical and XPS studies presented here do not show a clear correlation between electrochemical extent of oxidation (measured by the main PANI oxidation peak) and the doping level of the different PANI layers. The reason should be sought in the fact that the oxidation state of PANI, corresponding to the XPS measurements, does not necessarily coincide with the state obtained right after the leucoemeraldine to emeraldine electrochemical oxidation peak. This issue should be further investigated by taking into account also the possibility for a lower (than 100%) efficiency of the polymerization process that may affect the Finally, it is worth emphasizing that complete de-hydration of the polyacid-doped PANI layers results in re-ordering of the interpolymer complex structure of all four types of PANI materials. Emerging of sharp voltammetric oxidation peaks with perfect reversibility and stability is observed together with reversible dopant-dependent exchange of mass (ingress and egress of water molecules) upon voltammetric redox switching. All four types of PANI layers are found to preserve their electroactivity under long-term storage in the dry state.


It was observed that following a 24 h chilling treatment, slsbpase mutant plants accumulated higher levels of reactive oxygen species (ROS) than wild-type plants and consequently, more severe lipid peroxidation occurred in slsbpase plants. Activity assay of antioxidant enzymes showed that mutation in SlSBPASE significantly decreased activities of peroxidase (POD) and ascorbate peroxidase (APX), but surprisingly did not significantly alter activities of superoxide dismutase (SOD) and catalase (CAT) under the chilling condition. Notably, mutation in SlSBPASE reduced the contents of total ascorbate (AsA) and total glutathione (GSH) and suppressed the recycling of AsA and GSH in chilling-stressed tomato plants. In addition, activities of two GSH biosynthetic enzymes (gamma-glutamylcysteine synthetase and glutathione synthetase) and transcript abundance of their coding genes (GSH1 and GSH2) were markedly reduced in slsbpase mutant plants in comparison with those in wild-type plants under chilling stress. Furthermore, exogenous GSH remarkably mitigated chilling damage in slsbpase plants. Collectively, these results support that mutation in SlSBPASE aggravates chilling-induced oxidative stress by suppressing GSH biosynthesis and AsA-GSH recycling and suggest that SBPase is required for optimal response to chilling stress in tomato plants. The findings also shed light on the idea to mitigate chilling-induced damages by genetically manipulating a photosynthetic enzyme in plants.Sedoheptulose-1,7-bisphosphatase (SBPase) is a crucial enzyme for photosynthetic carbon assimilation in the Calvin-Benson cycle. Previous studies have shown that overexpression of SBPase is advantageous to chilling tolerance in plants; however, the mechanisms of SBPase acting in the improvement of chilling tolerance remain largely unknown. In the present study, we aimed to uncover the essential role of SBPase in the response of tomato plants to oxidative stress induced by low temperature. To fulfill that, we performed an array of comparative studies between  Cold stress impairs plant growth and development and severely reduces crop productivity . EffectsPlants also rely on the ascorbate-glutathione (AsA-GSH) cycle, which has been recognized as one effective antioxidant mechanism, to eliminate excessive ROS . The AsAChloroplasts harbor light-dependent reactions and carbon-fixing reactions of photosynthesis are considered as one of the major sources of ROS production under adverse conditions. Under low temperature stress, the electron transport chain is often over-reduced and electrons are passed on to molecular oxygen, which gives rise to the formation of ROS . The CalSlSBPASE was induced by low temperature and its overexpression enhanced tomato growth and chilling tolerance  that we previously generated (Solanum lycopersicum L. “Micro-Tom”) were used as plant materials in this study. In our previous study, we screened one homozygous mutant line with 1-bp deletion in the second exon of SlSBPASE and the transgenic line showed just residual activity of SBPase . The measurements were conducted under the controlled condition, with light density being 600 μmol mThe third leaves were detached from different groups of tomato plants at the end of day for extraction and measurement of sucrose. The measurements were conducted as described previously .2O2 accumulation and O2·− production. Quantification of H2O2 was conducted by recording the absorbance of Ti-H2O2 complex at 410 nm as described previously  level. Determination of MDA content was done as in a previous work . BrieflyFollowing a 24 h chilling treatment, leaves of wild-type plants and mutant plants were detached for measurement of electrolyte leakage as previously described .Leaf samples were collected after a 24 h chilling treatment and the enzymes were extracted with potassium phosphate buffer . After centrifugation, the supernatant was used to determine the antioxidant enzyme activities . To deteGSH1 and GSH2 gene transcript abundance was investigated by quantitative real-time PCR. Total RNA was extracted from different leaf samples and was used for cDNA synthesis. The determination was performed using a Premix Ex Taq kit  according to the manufacturer’s instructions. Each real-time PCR reaction was performed on iQ5 Multicolor Real-Time PCR Detection System . The primers used for GSH1 were 5'-CCTCAGCACACCAAAATCCT-3' (forward) and 5'-GCTTTGTGCCTTGGCTCTAGT-3' (reverse). The primers used for GSH2 were 5-AGTGGAAAGCTAGGCTGCTG-3' (forward) and 5'-TCATCCAAGCTCCACAACCC-3' (reverse).4. After centrifugation, the absorbance of the supernatant was measured at 265 nm in sodium acetate buffer and the absorbance of that solution following incubation with AsA oxidase (0.5 units) was also recorded at 265 nm. The difference in the absorbance was used to calculate AsA content. Measurement of GSH and GSSG contents was done as previously described  mutation in SlSBPASE increased ROS production and exacerbated lipid peroxidation in chilling-stressed tomato plants, (2) mutation in SlSBPASE negatively affected the AsA-GSH cycle under chilling stress, (3) mutation in SlSBPASE impairs GSH biosynthesis in chilling-stressed tomato plants, and (4) exogenous GSH partially recovers chilling resistance of slsbpase mutant plants.SBPase functions in the essential photosynthetic carbon assimilation pathway, Calvin-Benson cycle, in C3 plants. SBPase catalyzes the production of sedoheptulose-7-phosphate, which is further utilized to regenerate RuBP, the COd growth . Early wd growth ,b. In oud growth . Howevert plants . In this2O2 and O2·− in both mutant and wild-type tomato plants. However, the levels of ROS in slsbpase mutant plants were significantly higher than those in wild-type plants, suggesting that mutation in SlSBPASE increases ROS production. Photosynthesis takes place in chloroplast, which serves as a major site of ROS production , which is further converted to dehydroascorbate (DHA) by MDHAR. AsA is regenerated from its oxidized form DHA by DHAR and this reaction requires GSH as reducing agent (SlSBPASE significantly decreased activities of the four enzymes, which may ultimately lead to the decline in AsA and GSH as well as ratios of AsA/DHA and GSH/GSSG. These results partly explain the increased ROS accumulation in slsbpase mutant plants and substantiate the idea that SBPase favors removal of ROS via the AsA-GSH cycle.The ratios of AsA/DHA and GSH/GSSG determine the redox potentials of AsA and GSH. Relative to DHA and GSSH, higher levels of AsA and GSH are important for ROS detoxification in plants under stress conditions. AsA and GSH are connected in the AsA-GSH pathway and are recycled ng agent . AnotherSlSBPASE aggravates chilling-induced accumulation of ROS through impaired GSH biosynthesis and suppressed AsA-GSH recycling (SlSBPASE enhances chilling-induced oxidative stress in tomato plants, however, the impact of SlSBPASE overexpression on GSH biosynthesis has not been investigated. Thus, future studies should explore the relationship between overexpressing SlSBPASE and GSH biosynthesis under cold stress. The results may help increase our understanding of the mode of action of SlSBPASE in the tolerance to cold-induced oxidative stress in plants.In summary, our study has demonstrated a central role of SBPase in the mitigation of oxidative stress triggered by low temperature in tomato plants. Mutation in ecycling . Our preThe original contributions presented in the study are included in the article/supplementary material and further inquiries can be directed to the corresponding authors.SZ and FD designed the study. MW and FD performed the experiments and analyzed the data. MW, SZ, and FD discussed the results. MW drafted the manuscript and FD and SZ revised it. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.