Protective role of Chlorella vulgaris with Thiamine against Paracetamol induced toxic effects on haematological, biochemical, oxidative stress parameters and histopathological changes in Wistar rats

Paracetamol is extensively consumed as an analgesic and antipyretic drug, but at a high dose level, it leads to deleterious side effects, such as hepatic and nephrotoxicity. This research aimed to estimate the prophylactic efficacy of Chlorella vulgaris and/or thiamine against paracetamol (P) induced hepatorenal and cardiac toxicity. Forty-eight female Wistar rats were randomly divided into eight equal groups (n = 6 rats). Group 1, normal control group. Group 2, Paracetamol group. Groups 3, 4 and 5 were treated with Silymarin drug, Chlorella vulgaris alga, Chlorella vulgaris alga supplemented with thiamine, respectively daily for 7 successive days, then all were administered Paracetamol (2gm/kg. bwt.). While, Groups 6, 7 and 8 were treated by Silymarin, Chlorella vulgaris alga, Chlorella vulgaris supplemented with thiamine, respectively daily for 7 successive days without paracetamol administration. Our results clarified that Paracetamol toxicity caused significant adverse effects on hematological, serum biochemical parameters, and oxidant -antioxidant status as well as histopathological picture of heart, liver, and kidney. However, in the Paracetamol intoxicated groups pretreatment either with Chlorella vulgaris alone or plus thiamine successfully improved the undesirable deleterious effects of paracetamol, and restored almost all variables to near their control levels. This study has finished to that oxidative stress participates in the pathogenesis of paracetamol-induced toxicity in rats and using Chlorella vulgaris alga either alone or plus thiamine alongside their health benefits can protect against oxidative harmful effects induced by paracetamol through their free radical scavenging and powerful antioxidant effects, and they can be used as propylactic agents against paracetamol-induced toxicity.


Parameter group Final body wt
Serum biochemical parameters. The influences of paracetamol induced toxicity and the protective effects of C. vulgaris and /or thiamine on serum biochemical parameters are shown in (Figs. 1A,B, 2A,B). Paracetamol exposed rats group (G2) revealed significantly increased serum transaminases activities (Fig. 1A), cholesterol, bilirubin levels ( Fig. 2A) as well as elevated urea, and creatinine levels ( Fig. 2B) with significant decline in serum total protein and albumin concentrations (Fig. 1B) in comparison with normal control rats group (G1). Moreover, a significant alleviation in the same parameters was seen in G3, G4 and G5 compared with paracetamol exposed group (G2), the best improvement was observed in G3 and G5. However, a significant decline in ALT activity was shown in G6, G7 and G8 compared with normal control rat (G1). Meanwhile, a significant reduction in cholesterol was seen in G7 and G8 in comparison to normal control rat group (G1).
Hepatic renal and cardiac antioxidant status and lipid peroxidation. The influences of paracetamol induced toxicity and administration of C. vulgaris and /or thiamine on the lipid perioxidation and antioxidant enzymes of hepatic, renal, and cardiac tissues are shown in (Fig. 3A,B). MDA concentrations in hepatic, renal, and cardiac tissues were significantly elevated in paracetamol intoxicated group (G2) in comparison with the normal control group (G1) (Fig. 3A). Moreover, a significant decrease in hepatic, renal and cardiac MDA was observed in G3, G4, and G5 compared with paracetamol intoxicated group (G2). The superior reduction was observed in G3 and G5. On the other hand, paracetamol intoxication induced oxidative stress in liver, kidney and heart which resulted in the depletion of hepatic, renal and cardiac CAT activity (Fig. 3B). Furthermore, a significant elevation in catalase enzyme activity was detected in G3, G4 and G5 compared with paracetamol intoxicated group (G2), and the best was G5. Meanwhile, a significant increase in catalase activity was noticed in hepatic, renal, and cardiac tissues of rat groups administered C. vulgaris plus thiamine (G8) group compared to the normal control rat group (G1). vulgaris and thiamine when they were administered separately. Moreover, the paracetamol intoxicated group (Fig. 4E) revealed severe congestion, and most of the centrilobular hepatocytes showed marked vacuolar and ballooning degeneration, besides aggregation of lymphocytes in the portal area. The hepatic structure was improved and looked close normal with mild hydropic degeneration in hepatocytes in silymarin + paracetamol group and C. vulgaris + thiamine + paracetamol group (Fig. 4F,H). Moreover, paracetamol intoxicated rats given C. vulgaris showed moderate congestion, vacuolar and ballooning degeneration in hepatocytes (Fig. 4G). Kidney sections showed normal appearance of the glomerulus and tubules of control group (Fig. 5A). The same picture was seen in silymarin, C. vulgaris and C. vulgaris + thiamine treated groups, respectively ( Fig. 5B-D), confirming the nephroprotective effects of silymarin, C. vulgaris, and thiamine when given separately. Paracetamol intoxicated group (Fig. 5E) showed severe congestion, marked tubular dilation with loss of cellular boundary and epithelial degeneration , glomerular shrinkage, bleeding and partial endothelial rupture in capsule. Silymarin and C. vulgaris + Thiamine administrations to paracetamol intoxicated groups revealed mild congestion (Fig. 5F,H). While, C. vulgaris + paracetamol group was showed the moderate congestion beside the moderate tubular dilation as in (Fig. 5G).
Heart sections showed the normal appearance of cardiomyocytes of control rat group (Fig. 6A). The same findings were detected in silymarin, C. vulgaris and C. vulgaris + thiamine treated groups, respectively ( Fig. 6B-D). On the other hand, the paracetamol intoxicated group showed degeneration and vacuolation in cardiomyocytes with severely congested cardiac blood vessels (Fig. 6E). This lesion was much improved by the administration of either Silymarin or C. vulgaris plus Thiamine to the paracetamol intoxicated groups which showed mild congestion, respectively (Fig. 6F,H). While moderate congestion was seen by C. vulgaris administration to paracetamol intoxicated group (Fig. 6G). The infra-red spectrum displays a frequency ranges from 3500 to 3200 cm −1 indicating the O-H stretching vibration, existence of alcohols, phenols. The frequency ranges from, 3000-2850 cm −1 peaks are representing in the C-H stretching vibration existence of alkenes. The wavenumber of some peaks of C. vulgaris biomass were decreased or increased after supplemented with thiamine such as wavenumber peak at 3404 increased to 3449, the peak 2970 decreased to 2959, peaks at 2925 increased to 2954, peak 2856 decreased to 2853, peak 1655 decreased to 1646, peak 1054 increased to 1076 respectively. There are some new peaks and also some peaks are disappeared as shown in Table 5, these results showed the difference in the alga compositions when supplemented with thiamine and hence its effect on oxidative stress induced by paracetamol. Oxidative stress is a phenomenon caused by an imbalance between production and accumulation of oxygen reactive species (ROS) in cells and tissues and the ability of a biological system to detoxify these reactive products. ROS can play, and in fact they do it, several physiological roles (i.e., cell signaling), and they are normally generated as by-products of oxygen metabolism; despite this, environmental stressors (i.e., UV, ionizing radiations, pollutants, and heavy metals) and xenobiotics (i.e., antiblastic drugs) contribute to greatly increase ROS production, therefore causing the imbalance that leads to cell and tissue damage (oxidative stress) 32 .
Oxidative stress plays a vital role in the pathogenesis of paracetamol induced liver damage 33 . This study demonstrated that paracetamol intoxication caused deleterious impacts on hemopoietic organs, which represented by lowered hematological parameters including, RBCs counts, Hb concentration, PCV%, TLC, Platelets count and neutrophil%. These findings are consistent with that of Desnoyers 34 ;Taylor & Dhupa 35 who demonstrated that the changes in the analyzed blood parameters might be due to the oxidative stress induced by paracetamol which has a damaging effect on immune and hemopoietic organs and erythrocytes. Paracetamol inhibits hemopoesis together with hematotoxicity, primarily methemoglobinemia and hemolytic anemia. This may be attributed to the destruction of RBCs by increased lipid peroxidation in cell membranes 36 . Moreover, uremia has a bad effect on  Moreover, the significant elevation in cholesterol level recorded after paracetamol administration may be due to the imbalance between the normal rates of lipid synthesis, utilization and secretion 47,48 or may be duo to inhibition of bile acid synthesis as recorded by previous studies [49][50][51] . The reduced serum total protein and albumin concentrations following paracetamol overdose exposure in this study resulting from disturbance of protein synthesis as a consequence of altered hepatic function as a result of inflammation 52 or due to nephrotoxicity which leads to leakage of albumin in urine with decreasing of serum albumin and total protein concentrations 53 .
Our study clearly demonstrates that acute acetaminophen toxicity enhanced renal MDA level, depleted the renal CAT antioxidant activity leading to elevated serum urea and creatinine levels, reduced total protein and deteriorated the renal architecture as confirmed by our histopathological observations. The end product of lipid peroxidation is MDA, which is recognized as the second messenger of free radicals. The high concentration of MDA in renal tissue denotes to renal toxicity 54 . Inconsistent with our results, Srinivasan et al 33 who reported that, increased ROS level and decreased enzymatic antioxidants considered as a mechanism by which several chemicals can induce nephrotoxicity leading to disturbance of cell membrane integrity. Paracetamol nephrotoxicity occurs as a result of its highly reactive metabolite-NAPQI-which acrylates proteins in the proximal tubule, initiating renal tubular cells death 55  Several herbal and plant extracts derived compounds served as alternative therapeutic agents to counteract the side effects of various drugs 59,60 .
In the current study silymarin succeeded to overcome the deleterious impacts of paracetamol intoxication on rat hematological, biochemical parameters and histopathological changes, reduced hepatic, renal and cardiac oxidative damage and enhanced hepatic, renal and cardiac antioxidants. In consistent with our results, Papackova et al 8 who pointed that the main actions of silymarin are scavenging of radical forms of oxygen and inhibition of peroxynitrite formation. Furthermore, Freitag et al 10 stated that, the prophylactic activity of silymarin against paracetamol-induced hepatotoxicity is generally attributed to its antioxidant and anti-inflammatory properties. Several studies about the standard drug silymarin found that silymarin offered protection against chemical hepatotoxins such as CCl4, ethanol, and paracetamol 61 . Moreover, Cacciapuoti et al 62 mentioned that silymarin is an effective remedy for decreasing hepatic steatosis in patients with non-alcoholic fatty liver disease. Silymarin was approved for the treatment of the hepatotoxic doses of paracetamol. Therefore, in this research we used silymarin as a standard control drug. www.nature.com/scientificreports/ Regarding to the effect of C. vulgaris algae on body weight, our results showed a significant increase in final body weight and body weight gains in response to C. vulgaris algae administration in comparison to the control and other treated groups. C. vulgaris is a rich source for chlorophyll pigment and vital amino acids; in addition to considerable quantities of calcium, phosphorus, iodine, manganese, iron and vitamins such as A, B1, B2, B3, B6, B12, C 67 and E 63 . In agreement with our results, Xu et al 64 who stated that C. vulgaris can be a useful choice as an additive for fish diets, they claimed that C. vulgaris could improve digestive the enzymes and enhance growth performance and immunity due to its high concentrations of the crude protein, polysaccharides, lipid, minerals and other bioactive components involved in many physiological activities. On the same line, Kang et al 65 concluded that Chlorella additions to the diets of broiler chicks improved body weight.
Concerning to the effects of C. vulgaris against paracetamol intoxication, the current results demonstrated that rats administered C. vulgaris at the chosen dose either alone or with thiamine succeeded to minimize the deleterious effects of paracetamol on rats' hematological, biochemical, antioxidant status and histopathological findings, suggested that C. vulgaris exhibits excellent hepato protective properties and has some role in maintaining the structural integrity of the hepatocellular membrane, thus preventing the enzymes leakage into the blood circulation, together with repairing of the hepatic tissue damage induced by paracetamol. This impact is in consistent with Ahmed and Khater 66 , Pawlikowska-Pawlega et al 67 who stated that serum levels of transaminases returned to normal with the healing of hepatic parenchyma and the regeneration of hepatocytes. Moreover, Rodriguez-Garcia and Guil-Guerrero 68 reported that Chlorella vulgaris exhibited antioxidative and hepatoprotective effects. Furthermore, Cheng et al 69 recorded that the possible mechanism for C. vulgaris protection may be attributed to its immunomodulatory potential, that may stimulate the lymphocytes propagation and phagocytic activities of macrophages, promote the expressions of cytokines, improve the NK cells cytotoxicity, and ameliorate the histological changes of the spleen.
Furthermore, C. vulgaris succeeded to restore the levels of urea, and creatinine close to normal values thus preventing the kidney from damage. These restorative effects of C. vulgaris over the serum clinical chemistry correlate with previous studies used C. vulgaris for treating oxidative stress 31 . On the same line several studies declared that Chlorella vulgaris administration provided protection against membrane fragility with anti-inflammatory, antihypertensive, and antioxidative activities 18,19 . As C. vulgaris microalgae, contains many valuable antioxidants as chlorophyll, carotenoids, astaxanthin, lutein and phycobili-proteins 17 , with the highest amount of chlorophyll than any known plant. Moreover, C. vulgaris prevented the lipid peroxidation in hepatic, renal and cardiac tissues. In addition to, its ability to abolish the toxic effect of paracetamol on the examined tissues through increasing the activities of antioxidant enzymes. The protective effects of C. vulgaris and its antioxidant activity are attributed to their content of phenolic compounds 70 as there is a close positive relationship or correlation between the quantity of these compounds in C. vulgaris extract and their antioxidant activities due to their redox properties that play a vital role in capturing and scavenging free radicals, oxygen suppression and peroxide decomposition [71][72][73] . Furthermore, C. vulgaris extract significantly decreased the degree of lipid peroxidation and TBARS level in leukocytes in comparison to Ganoderma lucidum extract in vitro location 74 . The same results were detected when C. vulgaris is supplemented alone or with thiamine. In agreement with our observation Zhou et al 28 who reported that thiamine can reduce oxidative stress. Furthermore, Asensi Fabado and Munne-Bosch 29 stated that, the antioxidant activities of thiamine can be indirect, by providing NADH and NADPH to the antioxidant network, or direct, by acting as an antioxidant.
The prophylactic effects of C. vulgaris against oxidative stress induced by paracetamol intoxication in our study could be due to inhibition of lipid peroxidation and scavenging of free radicals as its administration was responsible for the increased resistance against oxidative stress induced by paracetamol which consequently plays a fundamental role in the pathogenesis of paracetamol induced liver damage 33,52 . The elevated levels of MDA demonstrated in the present study are in accordance with those of other investigators who reported the association between paracetamol toxicity and MDA elevation 75 . Moreover, C. vulgaris and or thiamine prevented the lipid peroxidation in hepatic, renal and cardiac tissues and improved the activities of antioxidant enzymes in rats tissues, such effects could be the mechanisms of their hepatorenal protection. This is in agreement with the report of Sabiu et al 76 who stated that acetaminophen mediated hepatic oxidative insults in rats had induced significant decrease in the activities of antioxidant enzymes.
Compared with the standard drug silymarin, no significant differences were detected in the protection induced by silymarin treatment and C. vulgaris and /or thiamine treatment, suggesting that C. vulgaris either alone or with thiamine succeeded to prevent disruption of organs function by protecting the lipids from peroxidation by ROS under paracetamol toxicity and enhancing antioxidant enzymes activity.

Material and methods
Chemicals. Paracetamol tablets (each tablet contains 500 mg) was obtained from El-Nasr Pharmaceutical Chemicals Co., Egypt. Paracetamol was suspended in pathogen-free normal distilled water prior usage. Silymarin capsules (Legalon 140) each capsule contains 140 mg was purchased from Ced Pharmaceutical Co, Giza, Egypt.
The diagnostic kits used for assaying hepatic and kidney performance tests, the levels of lipid peroxidation and antioxidants were obtained from Bio-Diagnostic Co., Giza, Egypt. All other chemicals used throughout the experiments were of high analytical grade. Thiamine powder was obtained from El-Nasr Pharmaceutical Chemicals Co, Egypt. www.nature.com/scientificreports/ Chlorella vulgaris alga supplemented with Thiamine. Two hundred ml of the BG11 nutritive culture medium were prepared and supplemented with 0.08 mg/L vitamin B1 (thiamine), after sterilization C. vulgaris was inoculated. The culture was incubated at natural day light at temperature 30 ± 2 °C and shaken gently twice a day to avoid clumping and enhance the growth. After 15 days of incubations, the culture was centrifuged, washed by distilled water and dried through the hot air oven at 60 °C until the constant weight was obtained. The required daily dose from Chlorella powder and Chlorella supplemented with thiamine administered to the animals in this study were dissolved in sterilized distilled water to be in suspension format using Ultrasonic homogenizer sonication (Biologics Inc. USA manufacturer and leading innovator) 77 .

Chlorella vulgaris alga (CV). Chlorella vulgaris alga
FT-IR analysis. FTIR spectroscopy is a technique required to define cell contents of microalgae 78 . FTIR spectra illustrates the macromolecular composition of the algal biomass depending on the infrared absorption of functional groups. So, FTIR spectroscopy permits the revealing of changes in the relative abundance of organic compounds such as carbohydrate, lipid and protein. This technique was used in many studies to define the changes in the macromolecular composition of microalgae caused by nutrient stress [79][80][81][82] . The change of functional groups present in dry algal biomass control and that supplemented with Thiamine has been described by Fourier transform infrared (FTIR) spectroscopy according to methods of Jebsen 83 . Animals and experimental design. This study was approved by the Research Ethical Committee of the Genetic Engineering and Research Institute, Sadat City University, Egypt. Forty eight female albino rats of Wistar strain (130-150 g) were obtained from the Animal House of the Genetic Engineering and Research Institute, University of Sadat City, Egypt and housed in well-ventilated plastic cages. The diet and water were provided ad-libitum. All rats were housed under standard husbandry conditions (25 ± 2 °C temp, 60 ± 5% relative humidity and 12 h photoperiod). Rats were kept untreated for two weeks for acclimatization prior treatment and were weighed at the starting of research (initial weight). All animal handling procedures, sample collection and disposal were according to the regulation of Institutional Animal Care and Use Committee (IACUC), Genetic Engineering and Research Institute University of Sadat City, Egypt, under approval number (gebriUSC-009- [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19]. Forty eight female albino rats of Wistar strain were randomly divided into eight equal groups (n = 6 rats each) as the following: Group 1, Normal control group, it was administered distilled water only per os (0.5 ml/rat) daily for 7 successive days.
Group 2, Paracetamol group, it was treated as normal control group for 7 successive days, then given Paracetamol per os once (2gm/kg.bwt.). according to Sharoud 26  Rats of all the experimental groups were anaesthetized and euthanized after 24 h of the last treatment for samples collection.
Sampling. At the end of the experiment (24 h after paracetmol administration), rats in all groups were fasted overnight and weighed to calculate the final body weights and weight gain. Then, blood samples were obtained from each rat via retro orbital bleeding under light ether anaesthesia (Sigma Chem. Co., St Louis, Mo. U.S.A). Two blood samples were taken from each rat. One sample was put into a tube containing heparin as anticoagulant for hematological assessment. The other sample was put in a tube without heparin and allowed to coagulate, then centrifuged at 3000 for 15 min. The clear sera were collected and kept at − 20 °C for subsequent biochemical analysis. After blood samples collection, rats were euthanized by cervical dislocation for tissue samples collection. Liver, kidney and heart from each rat were carefully excised, weighed and immediately cleaned with normal saline solution (0.9% NaCl). Each tissue sample was divided into two parts. A part was kept at − 80 °C for Malondialdehyde (MDA) and catalase (CAT) activity estimations. The other part was fixed in 10% neutral buffer formalin solution for further histopathological examinations.
Absolute and relative body and organ weights. Just before killings the rats at 8th day of experiment, final body weight of all rats in all experimental groups was calculated. The body gain was calculated from the difference between the body weight at the beginning and at the end of experiment. Upon being sacrificed or killed, the liver, kidney and heart were aseptically removed, weighed and their relative organ weights (ROW) were determined according to the equation of Aniagu et al 84  www.nature.com/scientificreports/ Hematological analysis. The whole blood samples were utilized directly after collection for estimation of hematological parameters including the red blood cells (RBCs), hemoglobin (Hb) concentration and hematocrit value (PCV%), total leucocytes count (TLC), differential leukocyte counts and platelets (Plt) counts, by using automated blood cells counter with an Auto Hematology Analyzer (Sysmex F-800, Japan) 85 .
Biochemical assays. The biochemical parameters of liver and renal injury biomarkers were estimated in the collected serum samples according to the manufacturer protocol. Serum enzymatic activities of aspartate amino transferase (AST) and alanine amino transferase (ALT) were assessed according to Reitman and Frankel 86 . Albumin (Alb) and total proteins (TP) according to Henry et al 87 . Serum cholesterol was measured according to Richmond 88 . Total bilirubin was analyzed according to Tietz 89 . Renal products; creatinine was estimated according to Larsen 90 , and urea according to Coulombe and Favreau 91 .

Evaluation of oxidative stress and antioxidant biomarkers.
Immediately after blood collection, the animals were euthanzied by cervical dislocation, then the liver, kidney and heart from each rat were immediately dissected out and weighed. A part from each organ was homogenized using glass homogenizer with ice cooled saline to prepare 25% W/V homogenate. This homogenate was centrifuged at 1700 rpm for 10 min; the supernatant was stored at -80 °C until analysis. This supernatant was used for the colorimetrical estimations of hepatic, renal and cardiac malondialdehyde (MDA), the main end product of lipid peroxidation, according to the protocol of Esterbauer et al 92 , and catalase activity according to the method of Sinha 93 .
Histopathological results. The other parts of liver, kidney and heart of the scarified rats were fixed in 10% buffered formalin. Then, dehydration, clearance and processing in paraffin were carried out. Tissue sectioning and staining with H&E were performed according to Bancroft et al 94 .
Statistical analysis. All data were expressed as means ± S.E. and statistically analyzed by one-way ANOVA and Tukey's post-hoc test multiple comparisons using Graphpad prism Version 5 software (Graph Pad Software Inc., USA).. Statistical significance was acceptable to a level of p ≤ 0.05.

Conclusions
Oxidative stress plays essential role in paracetamol induced hepatorenal and cardiac toxicity. C. vulgaris is a potent antioxidant agent that was indicated to protect intoxicated rats against oxidative stress induced by paracetamol. This study, revealed that paracetamol exposure resulted in varying degrees of lipid peroxidation, depletion of the antioxidant enzymes activity and changes of hematological, biochemical parameters and histopathological architectures of the examined tissues. C. vulgaris and /or thiamine pre-exposure offered near complete protection in terms of blood and tissues changes, antioxidant enzymes activity and oxidative stress. Therefore, this study suggested that C. vulgaris is a promizing protective agent against paracetamol induced toxicity as ROS scavenger and a potential source of natural antioxidants.