- Email: [email protected]
Protective effects of lycopene against AFB1-induced erythrocyte dysfunction and oxidative stress in mice
Research in Veterinary Science 129 (2020) 103–108
Contents lists available at ScienceDirect
Research in Veterinary Science journal homepage: www.elsevier.com/locate/rvsc
Protective effects of lycopene against AFB1-induced erythrocyte dysfunction and oxidative stress in mice
T
Jian Zhang1, Peiyan Wang1, Feibo Xu, Wanyue Huang, Qiang Ji, Yanfei Han, Bing Shao, ⁎ Yanfei Li Heilongjiang Key Laboratory for Laboratory Animals and Comparative Medicine, College of Veterinary Medicine, Northeast Agricultural University, Harbin 150030, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Lycopene Aflatoxin B1 Erythrocyte dysfunction Oxidative stress
To evaluate the protective role of lycopene (LYC) against aflatoxin B1 (AFB1)-induced erythrocyte dysfunction and oxidative stress, male kunming mice were treated with LYC (5 mg/kg) and/or AFB1 (0.75 mg/kg) by intragastric administration for 30 d. Hematological indices were detected to assess erythrocyte function. The erythrocytes C3b receptor rate (E-C3bRR) and erythrocytes C3b immune complex rosette rate (E-ICRR) were detected to assess erythrocyte immune function. Hydrogen peroxide (H2O2) and malondialdehyde (MDA) contents and superoxide dismutase (SOD) and catalase (CAT) activities were determined to evaluate erythrocyte oxidative stress. The results showed that LYC administration significantly relieved AFB1-induced erythrocyte dysfunction by increasing the levels of red blood cell count (RBC), hemoglobin (HGB) and hematocrit (HCT), as well as reducing red blood cell volume distribution width (RDW) level, while the levels of mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC) and mean platelet volume (MPV) had no significant differences among the four groups. Besides, LYC ameliorated AFB1-induced erythrocyte immune dysfunction by increasing E-C3bRR and decreasing E-ICRR. Furthermore, LYC also alleviated AFB1-induced erythrocyte oxidative stress by decreasing H2O2 and MDA contents and increasing SOD and CAT activities. These results indicated that LYC protected against AFB1-induced erythrocyte dysfunction and oxidative stress in mice. The findings could lead a possible therapeutics for the management of AFB1-induced erythrocyte toxicity.
1. Introduction Aflatoxins (AFs), secondary toxic fungal metabolites of Aspergillus flavus and Aspergillus parasiticus, are found in various agricultural commodities, particularly rice, corn and peanuts (Bennett and Klich, 2003). AFs-contaminated food occurs during production, harvest, transportation, storage and food processing (And and Gallagher, 1994). Globally, about 4.5 billion people are at risk of chronic exposure to AFs in developing countries (Williams et al., 2004). Also, AFs and its residues could be transferred to human being via food chain (Boonyaratpalin et al., 2001). Among the various AFs and their metabolites, aflatoxin B1 (AFB1) is the predominant form with the highest toxic potential and classified as group I cancerogen by the International Agency for Research on Cancer (Rothschild, 1992). AFB1 has hepatotoxic, immunosuppressive, carcinogenic, teratogenic and mutagenic effects (Dalel et al., 2011; Verma and Raval, 1991). Numerous epidemiological investigations confirm that the consumption of AFB1-
contaminated food directly depresses growth and health, inducing numerous metabolic disorder and hepatic disease (Gong et al., 2004; Groopman and Kensler, 2005; Lewis et al., 2005). Additionally, AFB1 has erythrocytic toxicity, causing erythrocyte hemolysis in cattle, guinea pigs, chicks and rabbits (Chung et al., 1985; Verma, 2004). Also, AFB1 can cause anemia in pregnant women, inducing intrauterine growth restriction, fetal loss, and preterm birth (Smith et al., 2017). The erythrocytic toxicity mechanism of AFB1 is still unclear. Erythrocytes are the most numerous cells in the blood, and the main medium for delivering oxygen to tissues and organs (Greer et al., 2008). Numerous studies reported AFB1 reduced the levels of red blood cell count (RBC), hemoglobin (HGB) and hematocrit (HCT) and impaired erythrocyte morphology, inhibiting the erythrocyte function and then causing anemia in humans and experimental animals (Tennant et al., 1974; Lanza et al., 1980). In addition, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), red blood cell volume distribution width
⁎
Corresponding author at: College of Veterinary Medicine, Northeast Agricultural University, NO. 600 Changjiang Road, Harbin 150030, China. E-mail address: [email protected] (Y. Li). 1 The two authors contributed equally to this study. https://doi.org/10.1016/j.rvsc.2020.01.015 Received 1 August 2018; Received in revised form 24 November 2019; Accepted 13 January 2020 0034-5288/ © 2020 Published by Elsevier Ltd.
Research in Veterinary Science 129 (2020) 103–108
J. Zhang, et al.
concentrations of 0.2 mg/mL and 1 mg/mL, respectively. The doses of AFB1 and LYC were selected based on the results of previous studies (Xu et al., 2017; Limpens et al., 2006; Lin et al., 2016), and the pre-experiments showed that 0.75 mg/kg AFB1 can induce erythrocyte dysfunction and oxidative stress in mice. The general health was monitored daily.
(RDW), platelet (PLT), mean platelet volume (MPV), platelet distribution width (PDW) and plateletcrit (PCT) were essential indexes to reflect the erythrocyte function. Meanwhile, erythrocytes are natural immunity cells. Siegel et al. (1981) firstly put forward the erythrocyte immunity, which can eliminate complement-opsonized circulatory immune complex (CIC), viruses, bacteria and tumor cells in vertebrates and is an important part of nonspecific immunity in vertebrates. Erythrocytes C3b receptor rate (E-C3bRR) and erythrocytes C3b immune complex rosette rate (E-ICRR) are important indicators reflecting the erythrocyte immune function. Wang et al. (2015) found that AFB1 induced erythrocyte immune dysfunction in chickens, which might be caused by oxidative stress. Oxidative stress is the pathological basis of AFB1-induced erythrocyte damage (Kanbur et al., 2011). AFB1-induced erythrocyte oxidative stress further impairs erythrocyte morphology, alters membrane permeability and releases intracellular HGB, which causes erythrocyte hemolysis and anemia (Kanbur et al., 2011; Yener et al., 2009). Altogether, antioxidants could be a potential therapeutic approach to prevent or slow the progression of AFB1-induced erythrocyte dysfunction and oxidative stress. Lycopene (LYC), a fat soluble naturally carotenoid pigment, is abundant in tomatoes, pink grapefruits and watermelons (Giovannucci, 1999). LYC is classified as food colorant by Joint FAO/WHO Expert Committee on Food Additives (Additives and Organization, 2000). Many epidemiological investigations have been reported LYC exerted the effects of anti-inflammatory, anticancer, anti-cardiovascular disease and detoxification (Herzog et al., 2005; Giovannucci et al., 2003; CostaRodrigues et al., 2018; Li et al., 2016). With a 40 carbon polyisoprenoid chain and 13 conjugated double bond structure, LYC is one of the most effective antioxidants in the carotenoid family (Bhuvaneswari and Nagini, 2005). LYC has strong scavenging activity of free radicals and defense capability of antioxidant system, protecting the liver, kidney, immune and nervous system against oxidative stress (Atessahin et al., 2005; Karahan et al., 2005; Ciçek et al., 2014; Porrini and Riso, 2000). LYC also protected liver and mucous membrane against AFB1-induced oxidative stress (Kurt et al., 2009; Xu et al., 2017). Besides, LYC is the potent antioxidant in human plasma (Mein et al., 2008), and protects against chlorpyrifos-induced erythrocyte function damage in carp (Ural, 2013). Based on these findings, we hypothesized that LYC could alleviate AFB1-induced erythrocyte dysfunction and oxidative stress. In the present study, the erythrocyte function (RBC, HGB, HCT, MCV, MCH, MCHC, RDW, PLT, MPV, PDW and PCT), erythrocyte immune function (E-C3bRR and E-ICRR) and erythrocyte oxidative stress (hydrogen peroxide (H2O2) and malondialdehyde (MDA) contents, superoxide dismutase (SOD) and catalase (CAT) activities) were detected in the present study to verify the hypothesis.
2.2. Blood sample collection The use of animals and the experimental protocol were approved by the Ethics Committee on the Use and Care of Animals, Northeast Agricultural University, China. All mice received humane care according to the criteria outlined in the “Guide for the Care and Use of Laboratory animals”. After 30 d, the mice were sacrificed under light ether anaesthesia and the blood samples were withdrawn from the retroorbital venous plexus by EDTA-containing tubes. Each blood sample was divided into 2 parts: one part was used to determine the hematological indices immediately; the other part was used for determination of erythrocyte immune function and oxidative stress. 2.3. Hematological indices analysis The following estimations were performed immediately with the help of a fully automatic hematology analyzer (BC-5000 Vet Mindary, China): RBC, HGB, HCT, MCV, MCH, MCHC, RDW, PLT, MPV, PDW and PCT. 2.4. Erythrocyte immune function assay The erythrocyte immune function was measured by erythrocyte yeast rosette formation test. The examination of E-C3bRR and E-ICRR were determined using the method described by Zhao et al. (2014). 2.5. Determination of oxidative stress 2.5.1. Preparation of erythrocytes and haemolysis The blood samples were centrifuged at 1000g for 5 min, and erythrocytes were separated from the plasma. Erythrocytes were washed thrice in phosphate buffered saline and diluted with an equal volume of this solution. A 0.4 mL portion of the washed erythrocytes was subjected to haemolysis by mixing with 1.6 mL of ice-cold water. 2.5.2. HGB estimation Erythrocyte hemoglobin concentration was determined using the commercial kit (C021 Nanjing Jiancheng Bioengineering Institute, China), and using cyanide methemoglobin method by a spectrophotometer (Gene Quant 1300 GE, USA).
2. Materials and methods
2.5.3. Determination of erythrocyte H2O2 and MDA contents Erythrocyte H2O2 and MDA contents were measured using the commercial kits (A064, A003–1, Nanjing Jiancheng Bioengineering Institute, China) following the manufacturer's instructions. H2O2 and MDA content were detected using thiobarbituric acid method and visible light method by a spectrophotometer (Gene Quant 1300 GE, USA). All experiments were repeated three times. H2O2 content was expressed as mmol/mL Hb and MDA content was expressed as nmol/mL Hb.
2.1. Animals and experimental design Forty healthy male kunming mice (6 weeks old) weighing 23–28 g were housed in the Biomedical Research Center, Northeast Agriculture University, China. The housing conditions were maintained at temperature of 24 ± 1 °C, relative humidity at 55 ± 5%, ventilation frequency at 18 times/h and a 12-h light/dark cycle. All mice were provided standard pellet diet (Xietong Organism, China) and distilled water ad libitum. After an acclimatization period of a week, the mice were divided into four groups, ten in each. The mice were treated for 30 days as follows: Group 1 (CG) was orally administered with the vehicle (0.2 mL olive oil). Group 2 (AG) was orally administrated with AFB1 (≥ 99.8%, Qingdao Pribolab Pte. Ltd., China) at 0.75 mg/kg. Group 3 (ALG) were treated with LYC (≥ 98%, Solarbio, Beijing, China) at 5 mg/kg 2 h prior to AFB1 administration, then given AFB1 at 0.75 mg/kg. Group 4 (LG) was orally administrated with LYC at 5 mg/kg. AFB1 and LYC were administered by gavage in olive oil and made into suspension at the
2.5.4. Determination of erythrocyte SOD and CAT activities Erythrocyte SOD and CAT activities were measured using the commercial kits (A001–3, A007–1, Nanjing Jiancheng Bioengineering Institute, China) following the manufacturer's instructions. SOD and CAT activities were detected using thiobarbituric acid method and visible light method by a spectrophotometer (Gene Quant 1300 GE, USA). All experiments were repeated three times. SOD activity was expressed as U/mgHb and CAT activity was expressed as U/mgHb. 104
Research in Veterinary Science 129 (2020) 103–108
J. Zhang, et al.
Table 1 Effects of LYC on hematological indices in AFB1-exposed mice. CG 12
RBC(10 /L) HGB(g/L) HCT(percent) MCV(μm3) MCH(pg) MCHC(g/L) RDW (percent) PLT(109/L) MPV(μm3) PDW(percent) PCT(percent)
9.39 ± 0.35 154.00 ± 3.74 49.25 ± 1.35 51.23 ± 1.38 16.20 ± 1.04 316.33 ± 19.60 13.33 ± 1.03 615.33 ± 5.99 6.77 ± 0.16 15.65 ± 0.29 0.75 ± 0.02
AG
ALG ⁎
8.50 ± 0.76 137.67 ± 3.61⁎⁎ 43.92 ± 1.16⁎⁎ 50.17 ± 2.04 15.08 ± 0.96 299.50 ± 23.95 15.45 ± 0.83⁎⁎ 369.50 ± 4.76⁎⁎ 6.18 ± 0.12 15.13 ± 0.12 0.54 ± 0.01
LG #
9.32 ± 0.45 143.00 ± 2.97⁎⁎,# 46.66 ± 1.12⁎⁎,## 50.23 ± 1.36 15.93 ± 0.70 317.50 ± 18.96 14.10 ± 0.58## 432.00 ± 8.99⁎⁎,## 6.40 ± 0.14 15.23 ± 0.14 0.74 ± 0.01
9.41 ± 0.64 153.67 ± 3.61 45.61 ± 2.28 51.57 ± 1.52 16.08 ± 0.85 311.83 ± 16.76 12.37 ± 0.59 612.33 ± 3.44 6.61 ± 0.16 15.63 ± 0.19 0.73 ± 0.01
All data were expressed as means ± SD (n = 6). ⁎p < .05 and ⁎⁎p < .01 symbol for the significance of differences versus the CG. #p < .05 and ##p < .01 symbol for the significance of differences versus the AG. CG control group, AG AFB1 group, ALG AFB1 + LYC group and LG LYC group.
when compared to the CG (p ≤ .01), whereas they were significantly reserved in ALG with respect to AG (p ≤ .01) (Fig. 2).
2.6. Statistical analysis Results are expressed as mean ± standard deviation (SD). The results were analyzed by one-way analysis of variance followed by Bonferroni's test (SPSS 22.0 software; SPSS Inc., Chicago, IL, USA). We considered p values of < 0.05 as significant and < 0.01 as markedly significant.
3.4. Effects of LYC on erythrocyte SOD and CAT activities in AFB1-exposed mice
3. Results
The SOD and CAT activities were significantly decreased in AG when compared to the CG (p ≤ .01), whereas they were significantly reserved in ALG with respect to AG (p ≤ .01) (Fig. 3).
3.1. Effects of LYC on hematological indices in AFB1-exposed mice
4. Discussion
The levels of RBC, HGB, HCT and PLT were significantly decreased and RDW level increased in AG when compared to the CG (p ≤ .05, p ≤ .01), whereas they were significantly reserved in ALG with respect to AG (p ≤ .05, p ≤ .01). The levels of MCV, MCH, MCHC, MPV, PDW and PCT had no significant differences among four groups. Meanwhile, there were no significant differences in hematological indices between CG and LG (Table 1).
In the present study, LYC significantly relieved AFB1-induced erythrocyte dysfunction by increasing the levels of RBC, HGB and HCT, as well as reducing RDW level. Besides, LYC ameliorated AFB1-induced erythrocyte immune dysfunction by increasing E-C3bRR and decreasing E-ICRR. Moreover, LYC alleviated AFB1-induced erythrocyte oxidative stress by decreasing H2O2 and MDA contents and increasing SOD and CAT activities. Hematological indices reflect the physiological, pathological and nutritional status of animals exposed to toxicants (Toghyani et al., 2010). The hematological alterations occur earlier than clinical symptoms in chronic aflatoxicosis (Kececi et al., 1998). The levels of RBC, HGB and HCT reflect the erythrocyte function, which deliver oxygen to the tissues and provide energy to aerobic respiration and metabolism (Schaer et al., 2013; Walker et al., 1990). Below normal levels of RBC, HGB and HCT in blood result in anemia (Wintrobe, 1974; Tennant et al., 1974). Besides, RDW level reflects morphology and heterogeneity of erythrocytes. Some investigations reported that AFB1 impaired erythrocyte function and caused anemia in chicken, calve and rabbit
3.2. Effects of LYC on E-C3bRR and E-ICRR in AFB1-exposed mice E-C3bRR was significantly decreased and E-ICRR was significantly increased in AG when compared to the CG (p ≤ .01), whereas they were significantly reserved in ALG with respect to AG (p ≤ .05) (Fig. 1). 3.3. Effects of LYC on erythrocyte H2O2 and MDA contents in AFB1exposed mice The H2O2 and MDA contents were significantly increased in AG
Fig. 1. Effects of LYC on E-C3bRR and E-ICRR levels in AFB1-exposed mice. (A) E-C3bRR; (B) E-ICRR. All data were expressed as means ± SD (n = 6). ⁎⁎p < .01 symbol for the significance of differences versus the CG. #p < .05 and ##p < .01 symbol for the significance of differences versus the AG. CG control group, AG AFB1 group, ALG AFB1 + LYC group and LG LYC group. 105
Research in Veterinary Science 129 (2020) 103–108
J. Zhang, et al.
Fig. 2. Effects of LYC on erythrocyte H2O2 and MDA contents in AFB1-exposed mice. (A) H2O2; (B) MDA. All data were expressed as means ± SD (n = 6). ⁎⁎p < .01 symbol for the significance of differences versus the CG. ##p < .01 symbol for the significance of differences versus the AG. CG control group, AG AFB1 group, ALG AFB1 + LYC group and LG LYC group.
et al., 2011). According to our foregoing results, we deduced LYC may enhance the quantity and activity of CR1 by increasing numbers and protecting morphology of erythrocytes to ameliorate erythrocyte immune dysfunction induced by AFB1. Oxidative stress is the pathological basis of erythrocyte damage (Farag and Alagawany, 2017). Erythrocyte oxidative stress impairs the membrane proteins and lipids, and intracellular HGB, which induces the production of H2O2 (Nagababu et al., 2003). Excess H2O2 results in lipid peroxidation (Karahan et al., 2005). MDA, the end product of lipid peroxidation, induces erythrocyte membrane oxidative stress (Freeman and Crapo, 1981). Erythrocytes protect against oxidative stress via antioxidant defense system including antioxidant enzymes as SOD and CAT (Cheung et al., 2001). AFB1-induced erythrocyte oxidative stress promotes the generation of H2O2 and MDA, and impairs the defense capability of antioxidant system (Amstad et al., 1984; Yener et al., 2009). In our study, LYC administration significantly decreased the H2O2 and MDA contents and increased the SOD and CAT activities in ALG, indicating that LYC alleviated AFB1-induced erythrocyte oxidative stress. The similar anti-oxidative effect of LYC has been found in T-2 toxin and zearalenone-induced intoxication (Leal et al., 1999; Boeira et al., 2014). These evidences support our results. Besides, LYC protects against the oxidation of proteins, lipids, and DNA in vivo (Gupta and Kumar, 2002; Stahl and Sies, 2003). Therefore, we deduced LYC may protect against the oxidation of HGB and membrane, relieving AFB1induced erythrocyte dysfunction. Moreover, the decreased MDA content in ALG indicated LYC alleviated AFB1-induced erythrocyte membrane oxidative stress, which may enhance the quantity and activity of CR1 and then ameliorate erythrocyte immune dysfunction induced by
(Majid and Noosha, 2014; Naseer et al., 2016; Verma and Raval, 1992). In our study, LYC administration significantly increased the levels of RBC, HGB and HCT, as well as decreased RDW level, indicating that LYC relieved the impairment of erythrocyte morphology and function, and anemia induced by AFB1. Numerous studies reported that AFB1 induced liver injury (Groopman and Kensler, 2005; Yener et al., 2009). Liver injury reduces hematopoietic factors (iron, folic acid and vitamin B12) and inhibits the synthesis of erythropoietin, subsequently reducing erythrocyte production (Radhakrishnan et al., 2013). LYC protected against AFB1-induced liver injury by enhancing detoxification and inhibiting the metabolism of AFB1 (Tang et al., 2007; Xu et al., 2017). Moreover, LYC acts as an effective erythroprotective agent by preventing malathion-induced toxic effects in crap and P. falciparum infection in human (Yonar, 2013; Agarwal et al., 2014). Thus, we deduced LYC may relieve AFB1-induced erythrocyte dysfunction via protecting erythrocytes, promoting detoxification in liver and up-regulation of hematopoietic factors in mice. Erythrocyte immune function can clear CIC, promote phagocytosis, present antigens and activate complement, which is mainly achieved by erythrocytes complement receptor type one (CR1). CR1 binds to CIC, viruses, bacteria and tumor cells, and prevents spreading to susceptible tissues, which reflects on E-C3bRR and E-ICRR (Garratty, 2010). EC3bRR represents the quantity and activity of CR1, and E-ICRR represents the content of CIC (Paccaud et al., 1990). In our study, LYC administration significantly increased E-C3bRR and decreased E-ICRR, indicating that LYC ameliorated AFB1-induced erythrocyte immune dysfunction. As CR1 exists on the erythrocyte membrane, erythrocyte membrane damage might reduce the quantity and activity of CR1 (Zhu
Fig. 3. Effects of LYC on erythrocyte SOD and CAT activities in AFB1-exposed mice. (A) SOD; (B) CAT. All data were expressed as means ± SD (n = 6). ⁎⁎p < .01 symbol for the significance of differences versus the CG. ##p < .01 symbol for the significance of differences versus the AG. CG control group, AG AFB1 group, ALG AFB1 + LYC group and LG LYC group. 106
Research in Veterinary Science 129 (2020) 103–108
J. Zhang, et al.
AFB1. LYC also protects lymphocyte against oxidative stress, improving the immune function (Böhm et al., 1995; Yonar, 2012). Thus, we deduced that the protective effects of LYC on erythrocyte immune dysfunction induced by AFB1 are associated with alleviating oxidative stress. However, the limitation of this study is without researching the mechanism of LYC attenuates oxidative stress induced by AFB1, which needs to be elucidated in our further studies. Interestingly, we found that LYC administration significantly increased the PLT level, indicating that LYC may alleviate the coagulation dysfunction induced by AFB1. PLT is the fragments of cytoplasm and derives from the megakaryocytes of bone marrow (Everts et al., 2006). Li et al. found that LYC can protect bone marrow mesenchymal stem cells (MSCs) against ischemiainduced apoptosis in vitro (Li et al., 2014). The patent suggests that MSCs can be used for megakaryocytes production (Cheng et al., 2000). In light of these, we deduced that LYC may protect MSCs and promote megakaryocytes production to alleviate the coagulation dysfunction induced by AFB1. In addition, the effect of LYC on megakaryocytes production and coagulation function exposed AFB1 is worth further studying.
on the ototoxicity induced by cisplatin. Turk J Med Sci 44, 582–585. Costa-Rodrigues, J., Pinho, O., Prr, M., 2018. Can lycopene be considered an effective protection against cardiovascular disease? Food Chem. 245, 1148–1153. Dalel, B., Chayma, B., Yousra, A., 2011. Chemopreventive effect of cactusOpuntia ficus indicaon oxidative stress and genotoxicity of aflatoxin B1. Nutr Metab 8, 73. Everts, P.A., Knape, J.T., Weibrich, G., Schönberger, J.P., Hoffmann, J., Overdevest, E.P., Box, H.A., Van, Z.A., 2006. Platelet-rich plasma and platelet gel: a review. J Extra Corpor Technol 38, 174. Farag, M.R., Alagawany, M., 2017. Erythrocytes as a biological model for screening of xenobiotics toxicity. Chem. Biol. Interact. 279, 73. Freeman, B.A., Crapo, J.D., 1981. Hyperoxia increases oxygen radical production in rat lungs and lung mitochondria. J. Biol. Chem. 256, 10986. Garratty, G., 2010. Advances in red blood cell immunology 1960 to 2009. Transfusion 50, 526–535. Giovannucci, E., 1999. Response: re: tomatoes, tomato-based products, lycopene, and prostate cancer: review of the epidemiologic literature. J. Natl. Cancer Inst. 91, 1331. Giovannucci, E., Rimm, E.Y., Stampfer, M.J., Willett, W.C., 2003. A prospective study of cruciferous vegetables and prostate cancer. Cancer Epidem Biomar 12, 1403–1409. Gong, Y., Hounsa, A., Egal, S., Turner, P.C., Sutcliffe, A.E., Hall, A.J., Cardwell, K., Wild, C.P., 2004. Postweaning exposure to aflatoxin results in impaired child growth: a longitudinal study in Benin, West Africa. Environ Health Persp 112, 1334–1338. Greer, J.P., Arber, D.A., Rodgers, G.M., Paraskevas, F., Foerster, J., Glader, B.E., Means, R.T., 2008. Wintrobe's Clinical Hematology 2 vols 12e. Lippincott Williams & Wilkins. Groopman, J.D., Kensler, T.W., 2005. Role of metabolism and viruses in aflatoxin-induced liver cancer. Toxicol Appl Pharm 206, 131–137. Gupta, N.P., Kumar, R., 2002. Lycopene therapy in idiopathic male infertility—a preliminary report. Int. Urol. Nephrol. 34, 369–372. Herzog, A., Siler, U., Spitzer, V., Seifert, N., Denelavas, A., Hunziker, P.B., Hunziker, W., Goralczyk, R., Wertz, K., 2005. Lycopene reduced gene expression of steroid targets and inflammatory markers in normal rat prostate. FASEB J. 19, 272–274. Joint FAO/WHO Expert Committee on Food Additives, WH Organization, 2000. Evaluation of certain food additives. In: Fifty-First Report of the Joint FAO/WHO Expert Committee on Food Additives. World Health Organization. Kanbur, M., Eraslan, G., Sarica, Z.S., Aslan, O., 2011. The effects of evening primrose oil on lipid peroxidation induced by subacute aflatoxin exposure in mice. Food Chem. Toxicol. 49, 1960–1964. Karahan, I., Ateşşahin, A., Yilmaz, S., Ceribaşi, A.O., Sakin, F., 2005. Protective effect of lycopene on gentamicin-induced oxidative stress and nephrotoxicity in rats. Toxicology 215, 198–204. Kececi, T., Oguz, H., Kurtoglu, V., Demet, O., 1998. Effects of polyvinylpolypyrrolidone,synthetic zeolite and bentonite on serum biochemical and haematological characters of broiler chickens during aflatoxicosis. Br. Poult. Sci. 39, 452–458. Kurt, D., Saruhan, B.G., Yokus, B., Cakir, D.U., 2009. Effects of lycopene and vitamine E administration over gastric mucosal damage induced by aflatoxin B. Journal of Animal & Veterinary Advances 8, 1256–1262. Lanza, G.M., Washburn, K.W., Wyatt, R.D., 1980. Strain variation in hematological response of broilers to dietary aflatoxin. Poultry Sci 59, 2686–2691. Leal, M., Shimada, A., Ruíz, F., González, dM E., 1999. Effect of lycopene on lipid peroxidation and glutathione-dependent enzymes induced by T-2 toxin in vivo. Toxicol. Lett. 109, 1–10. Lewis, L., Onsongo, M., Njapau, H., Schurz-Rogers, H., Luber, G., Kieszak, S., Nyamongo, J., Backer, L., Dahiye, A.M., Misore, A., 2005. Aflatoxin contamination of commercial maize products during an outbreak of acute aflatoxicosis in eastern and central Kenya. Environ. Health Perspect. 113, 1763–1767. Li, W., Jiang, B., Cao, X., Xie, Y., Huang, T., 2016. Protective effect of lycopene on fluoride-induced ameloblasts apoptosis and dental fluorosis through oxidative stressmediated caspase pathways. Chem. Biol. Interact. 261, 27–34. Li, Y., Xue, F., Xu, S.Z., Wang, X.W., Tong, X., Lin, X.J., 2014. Lycopene protects bone marrow mesenchymal stem cells against ischemia-induced apoptosis in vitro. Eur. Rev. Med. Pharmacol. Sci. 18, 1625–1631. Limpens, J., Schröder, F.H., de Ridder, C.M., Bolder, C.A., Wildhagen, M.F., ObermüllerJevic, U.C., Krämer, K., van Weerden, W.M., 2006. Combined lycopene and vitamin E treatment suppresses the growth of PC-346C human prostate cancer cells in nude mice. J. Nutr. 136, 1287. Lin, J., Zhao, H.S., Xiang, L.R., Xia, J., Wang, L.L., Li, X.N., Li, J.L., Zhang, Y., 2016. Lycopene protects against atrazine-induced hepatic ionic homeostasis disturbance by modulating ion-transporting ATPases. J. Nutr. Biochem. 27, 249–256. Majid, G.A., Noosha, Z.J., 2014. Effect of nanosilver on blood parameters in chickens having aflatoxicosis. Toxicol. Ind. Health 30, 192–196. Mein, J.R., Lian, F., Wang, X.D., 2008. Biological activity of lycopene metabolites: implications for cancer prevention. Nutr. Rev. 66, 667–683. Nagababu, E., Chrest, F.J., Rifkind, J.M., 2003. Hydrogen-peroxide-induced heme degradation in red blood cells: the protective roles of catalase and glutathione peroxidase. Biochim. Biophys. Acta 1620, 211–217. Naseer, O., Khan, J.A., Khan, M.S., Omer, M.O., Chishti, G.A., Sohail, M.L., Saleem, M.U., 2016. Comparative efficacy of Silymarin and Choline Chloride (Liver Tonics) in preventing the effects of aflatoxin B1 in bovine calves. Pol. J. Vet. Sci. 19, 545–551. Paccaud, J.P., Carpentier, J.L., Schifferli, J.A., 1990. Difference in the clustering of complement receptor type 1 (CR1) on polymorphonuclear leukocytes and erythrocytes: effect on immune adherence. Eur. J. Immunol. 20, 283–289. Porrini, M., Riso, P., 2000. Lymphocyte lycopene concentration and DNA protection from oxidative damage is increased in women after a short period of tomato consumption. J. Nutr. 130, 189–192. Radhakrishnan, K., Bishop, J., Jin, Z., Kothari, K., Bhatia, M., George, D., Garvin Jr., J.H.,
5. Conclusions In summary, LYC exerts protective effects against AFB1-induced erythrocyte dysfunction and oxidative stress in mice. The findings indicate LYC can be used as a protective agent against AFB1-induced erythrocyte toxicity. Declaration of Competing Interest The authors declare no potential conflicts. Acknowledgments This work was supported by a research grant from the open project program of Northeastern Science Inspection Station, China Ministry of Agriculture Key Laboratory of Animal Pathogen Biology (No.DY201709). References Agarwal, S., Sharma, V., Kaul, T., Abdin, M.Z., Singh, S., 2014. Cytotoxic effect of carotenoid phytonutrient lycopene on P. falciparum infected erythrocytes. Mol. Biochem. Parasitol. 197, 15–20. Amstad, P., Levy, A., Emerit, I., Cerutti, P., 1984. Evidence for membrane-mediated chromosomal damage by aflatoxin B1 in human lymphocytes. Carcinogenesis 5, 719–723. And, D.L.E., Gallagher, E.P., 1994. Mechanisms of aflatoxin carcinogenesis. Annu. Rev. Pharmacol. Toxicol. 34, 135–172. Atessahin, A., Yilmaz, S., Karahan, I., Ceribasi, A.O., Karaoglu, A., 2005. Effects of lycopene against cisplatin-induced nephrotoxicity and oxidative stress in rats. Toxicology 212, 116–123. Bennett, J.W., Klich, M., 2003. Mycotoxins. Clin. Microbiol. Rev. 16, 497–516. Bhuvaneswari, V., Nagini, S., 2005. Lycopene: a review of its potential as an anticancer agent. Curr Med Chem Anticancer Agents 5, 627–635. Boeira, S.P., Filho, C.B., Del’Fabbro, L., Roman, S.S., Royes, L.F.F., Fighera, M.R., Jessé, C.R., Oliveira, M.S., Furian, A.F., 2014. Lycopene treatment prevents hematological, reproductive and histopathological damage induced by acute zearalenone administration in male Swiss mice. Exp. Toxicol. Pathol. 66, 179–185. Böhm, F., Tinkler, J.H., Truscott, T.G., 1995. Carotenoids protect against cell membrane damage by the nitrogen dioxide radical. Nat. Med. 1, 98–99. Boonyaratpalin, M., Supamattaya, K., Verakunpiriya, V., Suprasert, D., 2001. Effects of aflatoxin B1 on growth performance, blood components, immune function and histopathological changes in black tiger shrimp (Penaeus monodon Fabricius). Aquac. Res. 32, 388–398. Cheng, L., Qasba, P., Vanguri, P., Thiede, M.A., 2000. Human mesenchymal stem cells support megakaryocyte and pro-platelet formation from CD34(+) hematopoietic progenitor cells. J. Cell. Physiol. 184, 58–69. Cheung, C.C., Zheng, G.J., Li, A.M., Richardson, B.J., Lam, P.K., 2001. Relationships between tissue concentrations of polycyclic aromatic hydrocarbons and antioxidative responses of marine mussels, Perna viridis. Aquat. Toxicol. 52, 189–203. Chung, W.M., Lin, J.K., Wu, K.C., Hsiung, K.P., 1985. In vitro interconversion of aflatoxin B1 and aflatoxicol by rat erythrocytes. Biochem. Pharmacol. 34, 2566. Ciçek, M.T., Kalcioğlu, T.M., Bayindir, T., Toplu, Y., Iraz, M., 2014. The effect of lycopene
107
Research in Veterinary Science 129 (2020) 103–108
J. Zhang, et al.
aflatoxicosis in rabbits. Bull. Environ. Contam. Toxicol. 49, 861–865. Walker, H.K., Hall, W.D., Hurst, J.W., 1990. Clinical methods:the history, physical, and laboratory examinations. Journal of the American Medical Association 264 (21), 2808–2809. Wang, C.H., Peng, X., Cui, H.M., Fang, J., 2015. Effects of aflatoxin B1 on the erythrocyte count, the content of hemoglobin, and the immune adherence function of erythrocytes in chickens. Med Weter 71, 758–762. Williams, J.H., Phillips, T.D., Jolly, P.E., Stiles, J.K., Jolly, C.M., Aggarwal, D., 2004. Human aflatoxicosis in developing countries: a review of toxicology, exposure, potential health consequences, and interventions. Am. J. Clin. Nutr. 80, 1106–1122. Wintrobe, M.M., 1974. Clinical hematology. South. Med. J. 35, 780–782. Xu, F., Yu, K., Yu, H., Wang, P., Song, M., Xiu, C., Li, Y., 2017. Lycopene relieves AFB 1 -induced liver injury through enhancing hepatic antioxidation and detoxification potential with Nrf2 activation. J. Funct. Foods 39, 215–224. Yener, Z., Celik, I., Ilhan, F., Bal, R., 2009. Effects of Urtica dioica L. seed on lipid peroxidation, antioxidants and liver pathology in aflatoxin-induced tissue injury in rats. Food Chem. Toxicol. 47, 418–424. Yonar, M.E., 2012. The effect of lycopene on oxytetracycline-induced oxidative stress and immunosuppression in rainbow trout (Oncorhynchus mykiss, W.). Fish Shellfish Immun 32, 994–1001. Yonar, S.M., 2013. Toxic effects of malathion in carp, Cyprinus carpio carpio: protective role of lycopene. Ecotox Environ Safe 97, 223–229. Zhao, Y.Z., Jia, J., Li, Y.B., Guo, C.X., Zhou, X.Q., Sun, Z.W., 2014. Effects of endosulfan on the immune function of erythrocytes, and potential protection by testosterone propionate. J. Toxicol. Sci. 39, 701–710. Zhu, Y., Zhao, H., Li, X., Zhang, L., Hu, C., Shao, B., Sun, H., Bah, A.A., Li, Y., Zhang, Z., 2011. Effects of subchronic aluminum exposure on the immune function of erythrocytes in rats. Biol. Trace Elem. Res. 143, 1576–1580.
Martinez, M., Ovchinsky, N., Lobritto, S., Elsayed, Y., Satwani, P., 2013. Risk factors associated with liver injury and impact of liver injury on transplantation-related mortality in pediatric recipients of allogeneic hematopoietic stem cell transplantation. Biol Blood Marrow Transplant 19, 912–917. Rothschild, L.J., 1992. IARC classes AFB1 as class 1 human carcinogen. Food Chem. News 34, 62–66. Schaer, D.J., Buehler, P.W., Alayash, A.I., Belcher, J.D., Vercellotti, G.M., 2013. Hemolysis and free hemoglobin revisited: exploring hemoglobin and hemin scavengers as a novel class of therapeutic proteins. Blood 121, 1276–1284. Siegel, I., Liu, T.L., Gleicher, N., 1981. The red-cell immune system. Lancet 2, 556. Smith, L.E., Prendergast, A.J., Turner, P.C., Humphrey, J.H., Stoltzfus, R.J., 2017. Aflatoxin exposure during pregnancy, maternal anemia, and adverse birth outcomes. Am J Trop Med 96, 770–776. Stahl, W., Sies, H., 2003. Antioxidant activity of carotenoids. Mol. Asp. Med. 24, 345–351. Tang, L., Guan, H., Ding, X., 2007. Modulation of aflatoxin toxicity and biomarkers by lycopene in F344 rats. Toxicol Appl Pharm 219, 10–17. Tennant, B., Harrold, D., Reina-Guerra, M., Kendrick, J.W., Laben, R.C., 1974. Hematology of the neonatal calf: erythrocyte and leukocyte values of normal calves. Cornell Veterinarian 64, 516. Toghyani, M., Tohidi, M., Gheisari, A.A., Tabeidian, S.A., 2010. Performance, immunity, serum biochemical and hematological parameters in broiler chicks fed dietary thyme as alternative for an antibiotic growth promoter. Afr. J. Biotechnol. 9, 6819–6825. Ural, M.Ş., 2013. Chlorpyrifos-induced changes in oxidant/antioxidant status and haematological parameters of Cyprinus carpio carpio: ameliorative effect of lycopene. Chemosphere 90, 2059–2064. Verma, R.J., 2004. Aflatoxin cause DNA damage. Int. J. Hum. Genet. 4 (4), 231–236. Verma, R.J., Raval, P.J., 1991. Cytotoxicity of aflatoxin on red blood corpuscles. Bull. Environ. Contam. Toxicol. 47, 428–432. Verma, R.J., Raval, P.J., 1992. Alterations in erythrocytes during induced chronic
108
Contents lists available at ScienceDirect
Research in Veterinary Science journal homepage: www.elsevier.com/locate/rvsc
Protective effects of lycopene against AFB1-induced erythrocyte dysfunction and oxidative stress in mice
T
Jian Zhang1, Peiyan Wang1, Feibo Xu, Wanyue Huang, Qiang Ji, Yanfei Han, Bing Shao, ⁎ Yanfei Li Heilongjiang Key Laboratory for Laboratory Animals and Comparative Medicine, College of Veterinary Medicine, Northeast Agricultural University, Harbin 150030, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Lycopene Aflatoxin B1 Erythrocyte dysfunction Oxidative stress
To evaluate the protective role of lycopene (LYC) against aflatoxin B1 (AFB1)-induced erythrocyte dysfunction and oxidative stress, male kunming mice were treated with LYC (5 mg/kg) and/or AFB1 (0.75 mg/kg) by intragastric administration for 30 d. Hematological indices were detected to assess erythrocyte function. The erythrocytes C3b receptor rate (E-C3bRR) and erythrocytes C3b immune complex rosette rate (E-ICRR) were detected to assess erythrocyte immune function. Hydrogen peroxide (H2O2) and malondialdehyde (MDA) contents and superoxide dismutase (SOD) and catalase (CAT) activities were determined to evaluate erythrocyte oxidative stress. The results showed that LYC administration significantly relieved AFB1-induced erythrocyte dysfunction by increasing the levels of red blood cell count (RBC), hemoglobin (HGB) and hematocrit (HCT), as well as reducing red blood cell volume distribution width (RDW) level, while the levels of mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC) and mean platelet volume (MPV) had no significant differences among the four groups. Besides, LYC ameliorated AFB1-induced erythrocyte immune dysfunction by increasing E-C3bRR and decreasing E-ICRR. Furthermore, LYC also alleviated AFB1-induced erythrocyte oxidative stress by decreasing H2O2 and MDA contents and increasing SOD and CAT activities. These results indicated that LYC protected against AFB1-induced erythrocyte dysfunction and oxidative stress in mice. The findings could lead a possible therapeutics for the management of AFB1-induced erythrocyte toxicity.
1. Introduction Aflatoxins (AFs), secondary toxic fungal metabolites of Aspergillus flavus and Aspergillus parasiticus, are found in various agricultural commodities, particularly rice, corn and peanuts (Bennett and Klich, 2003). AFs-contaminated food occurs during production, harvest, transportation, storage and food processing (And and Gallagher, 1994). Globally, about 4.5 billion people are at risk of chronic exposure to AFs in developing countries (Williams et al., 2004). Also, AFs and its residues could be transferred to human being via food chain (Boonyaratpalin et al., 2001). Among the various AFs and their metabolites, aflatoxin B1 (AFB1) is the predominant form with the highest toxic potential and classified as group I cancerogen by the International Agency for Research on Cancer (Rothschild, 1992). AFB1 has hepatotoxic, immunosuppressive, carcinogenic, teratogenic and mutagenic effects (Dalel et al., 2011; Verma and Raval, 1991). Numerous epidemiological investigations confirm that the consumption of AFB1-
contaminated food directly depresses growth and health, inducing numerous metabolic disorder and hepatic disease (Gong et al., 2004; Groopman and Kensler, 2005; Lewis et al., 2005). Additionally, AFB1 has erythrocytic toxicity, causing erythrocyte hemolysis in cattle, guinea pigs, chicks and rabbits (Chung et al., 1985; Verma, 2004). Also, AFB1 can cause anemia in pregnant women, inducing intrauterine growth restriction, fetal loss, and preterm birth (Smith et al., 2017). The erythrocytic toxicity mechanism of AFB1 is still unclear. Erythrocytes are the most numerous cells in the blood, and the main medium for delivering oxygen to tissues and organs (Greer et al., 2008). Numerous studies reported AFB1 reduced the levels of red blood cell count (RBC), hemoglobin (HGB) and hematocrit (HCT) and impaired erythrocyte morphology, inhibiting the erythrocyte function and then causing anemia in humans and experimental animals (Tennant et al., 1974; Lanza et al., 1980). In addition, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), red blood cell volume distribution width
⁎
Corresponding author at: College of Veterinary Medicine, Northeast Agricultural University, NO. 600 Changjiang Road, Harbin 150030, China. E-mail address: [email protected] (Y. Li). 1 The two authors contributed equally to this study. https://doi.org/10.1016/j.rvsc.2020.01.015 Received 1 August 2018; Received in revised form 24 November 2019; Accepted 13 January 2020 0034-5288/ © 2020 Published by Elsevier Ltd.
Research in Veterinary Science 129 (2020) 103–108
J. Zhang, et al.
concentrations of 0.2 mg/mL and 1 mg/mL, respectively. The doses of AFB1 and LYC were selected based on the results of previous studies (Xu et al., 2017; Limpens et al., 2006; Lin et al., 2016), and the pre-experiments showed that 0.75 mg/kg AFB1 can induce erythrocyte dysfunction and oxidative stress in mice. The general health was monitored daily.
(RDW), platelet (PLT), mean platelet volume (MPV), platelet distribution width (PDW) and plateletcrit (PCT) were essential indexes to reflect the erythrocyte function. Meanwhile, erythrocytes are natural immunity cells. Siegel et al. (1981) firstly put forward the erythrocyte immunity, which can eliminate complement-opsonized circulatory immune complex (CIC), viruses, bacteria and tumor cells in vertebrates and is an important part of nonspecific immunity in vertebrates. Erythrocytes C3b receptor rate (E-C3bRR) and erythrocytes C3b immune complex rosette rate (E-ICRR) are important indicators reflecting the erythrocyte immune function. Wang et al. (2015) found that AFB1 induced erythrocyte immune dysfunction in chickens, which might be caused by oxidative stress. Oxidative stress is the pathological basis of AFB1-induced erythrocyte damage (Kanbur et al., 2011). AFB1-induced erythrocyte oxidative stress further impairs erythrocyte morphology, alters membrane permeability and releases intracellular HGB, which causes erythrocyte hemolysis and anemia (Kanbur et al., 2011; Yener et al., 2009). Altogether, antioxidants could be a potential therapeutic approach to prevent or slow the progression of AFB1-induced erythrocyte dysfunction and oxidative stress. Lycopene (LYC), a fat soluble naturally carotenoid pigment, is abundant in tomatoes, pink grapefruits and watermelons (Giovannucci, 1999). LYC is classified as food colorant by Joint FAO/WHO Expert Committee on Food Additives (Additives and Organization, 2000). Many epidemiological investigations have been reported LYC exerted the effects of anti-inflammatory, anticancer, anti-cardiovascular disease and detoxification (Herzog et al., 2005; Giovannucci et al., 2003; CostaRodrigues et al., 2018; Li et al., 2016). With a 40 carbon polyisoprenoid chain and 13 conjugated double bond structure, LYC is one of the most effective antioxidants in the carotenoid family (Bhuvaneswari and Nagini, 2005). LYC has strong scavenging activity of free radicals and defense capability of antioxidant system, protecting the liver, kidney, immune and nervous system against oxidative stress (Atessahin et al., 2005; Karahan et al., 2005; Ciçek et al., 2014; Porrini and Riso, 2000). LYC also protected liver and mucous membrane against AFB1-induced oxidative stress (Kurt et al., 2009; Xu et al., 2017). Besides, LYC is the potent antioxidant in human plasma (Mein et al., 2008), and protects against chlorpyrifos-induced erythrocyte function damage in carp (Ural, 2013). Based on these findings, we hypothesized that LYC could alleviate AFB1-induced erythrocyte dysfunction and oxidative stress. In the present study, the erythrocyte function (RBC, HGB, HCT, MCV, MCH, MCHC, RDW, PLT, MPV, PDW and PCT), erythrocyte immune function (E-C3bRR and E-ICRR) and erythrocyte oxidative stress (hydrogen peroxide (H2O2) and malondialdehyde (MDA) contents, superoxide dismutase (SOD) and catalase (CAT) activities) were detected in the present study to verify the hypothesis.
2.2. Blood sample collection The use of animals and the experimental protocol were approved by the Ethics Committee on the Use and Care of Animals, Northeast Agricultural University, China. All mice received humane care according to the criteria outlined in the “Guide for the Care and Use of Laboratory animals”. After 30 d, the mice were sacrificed under light ether anaesthesia and the blood samples were withdrawn from the retroorbital venous plexus by EDTA-containing tubes. Each blood sample was divided into 2 parts: one part was used to determine the hematological indices immediately; the other part was used for determination of erythrocyte immune function and oxidative stress. 2.3. Hematological indices analysis The following estimations were performed immediately with the help of a fully automatic hematology analyzer (BC-5000 Vet Mindary, China): RBC, HGB, HCT, MCV, MCH, MCHC, RDW, PLT, MPV, PDW and PCT. 2.4. Erythrocyte immune function assay The erythrocyte immune function was measured by erythrocyte yeast rosette formation test. The examination of E-C3bRR and E-ICRR were determined using the method described by Zhao et al. (2014). 2.5. Determination of oxidative stress 2.5.1. Preparation of erythrocytes and haemolysis The blood samples were centrifuged at 1000g for 5 min, and erythrocytes were separated from the plasma. Erythrocytes were washed thrice in phosphate buffered saline and diluted with an equal volume of this solution. A 0.4 mL portion of the washed erythrocytes was subjected to haemolysis by mixing with 1.6 mL of ice-cold water. 2.5.2. HGB estimation Erythrocyte hemoglobin concentration was determined using the commercial kit (C021 Nanjing Jiancheng Bioengineering Institute, China), and using cyanide methemoglobin method by a spectrophotometer (Gene Quant 1300 GE, USA).
2. Materials and methods
2.5.3. Determination of erythrocyte H2O2 and MDA contents Erythrocyte H2O2 and MDA contents were measured using the commercial kits (A064, A003–1, Nanjing Jiancheng Bioengineering Institute, China) following the manufacturer's instructions. H2O2 and MDA content were detected using thiobarbituric acid method and visible light method by a spectrophotometer (Gene Quant 1300 GE, USA). All experiments were repeated three times. H2O2 content was expressed as mmol/mL Hb and MDA content was expressed as nmol/mL Hb.
2.1. Animals and experimental design Forty healthy male kunming mice (6 weeks old) weighing 23–28 g were housed in the Biomedical Research Center, Northeast Agriculture University, China. The housing conditions were maintained at temperature of 24 ± 1 °C, relative humidity at 55 ± 5%, ventilation frequency at 18 times/h and a 12-h light/dark cycle. All mice were provided standard pellet diet (Xietong Organism, China) and distilled water ad libitum. After an acclimatization period of a week, the mice were divided into four groups, ten in each. The mice were treated for 30 days as follows: Group 1 (CG) was orally administered with the vehicle (0.2 mL olive oil). Group 2 (AG) was orally administrated with AFB1 (≥ 99.8%, Qingdao Pribolab Pte. Ltd., China) at 0.75 mg/kg. Group 3 (ALG) were treated with LYC (≥ 98%, Solarbio, Beijing, China) at 5 mg/kg 2 h prior to AFB1 administration, then given AFB1 at 0.75 mg/kg. Group 4 (LG) was orally administrated with LYC at 5 mg/kg. AFB1 and LYC were administered by gavage in olive oil and made into suspension at the
2.5.4. Determination of erythrocyte SOD and CAT activities Erythrocyte SOD and CAT activities were measured using the commercial kits (A001–3, A007–1, Nanjing Jiancheng Bioengineering Institute, China) following the manufacturer's instructions. SOD and CAT activities were detected using thiobarbituric acid method and visible light method by a spectrophotometer (Gene Quant 1300 GE, USA). All experiments were repeated three times. SOD activity was expressed as U/mgHb and CAT activity was expressed as U/mgHb. 104
Research in Veterinary Science 129 (2020) 103–108
J. Zhang, et al.
Table 1 Effects of LYC on hematological indices in AFB1-exposed mice. CG 12
RBC(10 /L) HGB(g/L) HCT(percent) MCV(μm3) MCH(pg) MCHC(g/L) RDW (percent) PLT(109/L) MPV(μm3) PDW(percent) PCT(percent)
9.39 ± 0.35 154.00 ± 3.74 49.25 ± 1.35 51.23 ± 1.38 16.20 ± 1.04 316.33 ± 19.60 13.33 ± 1.03 615.33 ± 5.99 6.77 ± 0.16 15.65 ± 0.29 0.75 ± 0.02
AG
ALG ⁎
8.50 ± 0.76 137.67 ± 3.61⁎⁎ 43.92 ± 1.16⁎⁎ 50.17 ± 2.04 15.08 ± 0.96 299.50 ± 23.95 15.45 ± 0.83⁎⁎ 369.50 ± 4.76⁎⁎ 6.18 ± 0.12 15.13 ± 0.12 0.54 ± 0.01
LG #
9.32 ± 0.45 143.00 ± 2.97⁎⁎,# 46.66 ± 1.12⁎⁎,## 50.23 ± 1.36 15.93 ± 0.70 317.50 ± 18.96 14.10 ± 0.58## 432.00 ± 8.99⁎⁎,## 6.40 ± 0.14 15.23 ± 0.14 0.74 ± 0.01
9.41 ± 0.64 153.67 ± 3.61 45.61 ± 2.28 51.57 ± 1.52 16.08 ± 0.85 311.83 ± 16.76 12.37 ± 0.59 612.33 ± 3.44 6.61 ± 0.16 15.63 ± 0.19 0.73 ± 0.01
All data were expressed as means ± SD (n = 6). ⁎p < .05 and ⁎⁎p < .01 symbol for the significance of differences versus the CG. #p < .05 and ##p < .01 symbol for the significance of differences versus the AG. CG control group, AG AFB1 group, ALG AFB1 + LYC group and LG LYC group.
when compared to the CG (p ≤ .01), whereas they were significantly reserved in ALG with respect to AG (p ≤ .01) (Fig. 2).
2.6. Statistical analysis Results are expressed as mean ± standard deviation (SD). The results were analyzed by one-way analysis of variance followed by Bonferroni's test (SPSS 22.0 software; SPSS Inc., Chicago, IL, USA). We considered p values of < 0.05 as significant and < 0.01 as markedly significant.
3.4. Effects of LYC on erythrocyte SOD and CAT activities in AFB1-exposed mice
3. Results
The SOD and CAT activities were significantly decreased in AG when compared to the CG (p ≤ .01), whereas they were significantly reserved in ALG with respect to AG (p ≤ .01) (Fig. 3).
3.1. Effects of LYC on hematological indices in AFB1-exposed mice
4. Discussion
The levels of RBC, HGB, HCT and PLT were significantly decreased and RDW level increased in AG when compared to the CG (p ≤ .05, p ≤ .01), whereas they were significantly reserved in ALG with respect to AG (p ≤ .05, p ≤ .01). The levels of MCV, MCH, MCHC, MPV, PDW and PCT had no significant differences among four groups. Meanwhile, there were no significant differences in hematological indices between CG and LG (Table 1).
In the present study, LYC significantly relieved AFB1-induced erythrocyte dysfunction by increasing the levels of RBC, HGB and HCT, as well as reducing RDW level. Besides, LYC ameliorated AFB1-induced erythrocyte immune dysfunction by increasing E-C3bRR and decreasing E-ICRR. Moreover, LYC alleviated AFB1-induced erythrocyte oxidative stress by decreasing H2O2 and MDA contents and increasing SOD and CAT activities. Hematological indices reflect the physiological, pathological and nutritional status of animals exposed to toxicants (Toghyani et al., 2010). The hematological alterations occur earlier than clinical symptoms in chronic aflatoxicosis (Kececi et al., 1998). The levels of RBC, HGB and HCT reflect the erythrocyte function, which deliver oxygen to the tissues and provide energy to aerobic respiration and metabolism (Schaer et al., 2013; Walker et al., 1990). Below normal levels of RBC, HGB and HCT in blood result in anemia (Wintrobe, 1974; Tennant et al., 1974). Besides, RDW level reflects morphology and heterogeneity of erythrocytes. Some investigations reported that AFB1 impaired erythrocyte function and caused anemia in chicken, calve and rabbit
3.2. Effects of LYC on E-C3bRR and E-ICRR in AFB1-exposed mice E-C3bRR was significantly decreased and E-ICRR was significantly increased in AG when compared to the CG (p ≤ .01), whereas they were significantly reserved in ALG with respect to AG (p ≤ .05) (Fig. 1). 3.3. Effects of LYC on erythrocyte H2O2 and MDA contents in AFB1exposed mice The H2O2 and MDA contents were significantly increased in AG
Fig. 1. Effects of LYC on E-C3bRR and E-ICRR levels in AFB1-exposed mice. (A) E-C3bRR; (B) E-ICRR. All data were expressed as means ± SD (n = 6). ⁎⁎p < .01 symbol for the significance of differences versus the CG. #p < .05 and ##p < .01 symbol for the significance of differences versus the AG. CG control group, AG AFB1 group, ALG AFB1 + LYC group and LG LYC group. 105
Research in Veterinary Science 129 (2020) 103–108
J. Zhang, et al.
Fig. 2. Effects of LYC on erythrocyte H2O2 and MDA contents in AFB1-exposed mice. (A) H2O2; (B) MDA. All data were expressed as means ± SD (n = 6). ⁎⁎p < .01 symbol for the significance of differences versus the CG. ##p < .01 symbol for the significance of differences versus the AG. CG control group, AG AFB1 group, ALG AFB1 + LYC group and LG LYC group.
et al., 2011). According to our foregoing results, we deduced LYC may enhance the quantity and activity of CR1 by increasing numbers and protecting morphology of erythrocytes to ameliorate erythrocyte immune dysfunction induced by AFB1. Oxidative stress is the pathological basis of erythrocyte damage (Farag and Alagawany, 2017). Erythrocyte oxidative stress impairs the membrane proteins and lipids, and intracellular HGB, which induces the production of H2O2 (Nagababu et al., 2003). Excess H2O2 results in lipid peroxidation (Karahan et al., 2005). MDA, the end product of lipid peroxidation, induces erythrocyte membrane oxidative stress (Freeman and Crapo, 1981). Erythrocytes protect against oxidative stress via antioxidant defense system including antioxidant enzymes as SOD and CAT (Cheung et al., 2001). AFB1-induced erythrocyte oxidative stress promotes the generation of H2O2 and MDA, and impairs the defense capability of antioxidant system (Amstad et al., 1984; Yener et al., 2009). In our study, LYC administration significantly decreased the H2O2 and MDA contents and increased the SOD and CAT activities in ALG, indicating that LYC alleviated AFB1-induced erythrocyte oxidative stress. The similar anti-oxidative effect of LYC has been found in T-2 toxin and zearalenone-induced intoxication (Leal et al., 1999; Boeira et al., 2014). These evidences support our results. Besides, LYC protects against the oxidation of proteins, lipids, and DNA in vivo (Gupta and Kumar, 2002; Stahl and Sies, 2003). Therefore, we deduced LYC may protect against the oxidation of HGB and membrane, relieving AFB1induced erythrocyte dysfunction. Moreover, the decreased MDA content in ALG indicated LYC alleviated AFB1-induced erythrocyte membrane oxidative stress, which may enhance the quantity and activity of CR1 and then ameliorate erythrocyte immune dysfunction induced by
(Majid and Noosha, 2014; Naseer et al., 2016; Verma and Raval, 1992). In our study, LYC administration significantly increased the levels of RBC, HGB and HCT, as well as decreased RDW level, indicating that LYC relieved the impairment of erythrocyte morphology and function, and anemia induced by AFB1. Numerous studies reported that AFB1 induced liver injury (Groopman and Kensler, 2005; Yener et al., 2009). Liver injury reduces hematopoietic factors (iron, folic acid and vitamin B12) and inhibits the synthesis of erythropoietin, subsequently reducing erythrocyte production (Radhakrishnan et al., 2013). LYC protected against AFB1-induced liver injury by enhancing detoxification and inhibiting the metabolism of AFB1 (Tang et al., 2007; Xu et al., 2017). Moreover, LYC acts as an effective erythroprotective agent by preventing malathion-induced toxic effects in crap and P. falciparum infection in human (Yonar, 2013; Agarwal et al., 2014). Thus, we deduced LYC may relieve AFB1-induced erythrocyte dysfunction via protecting erythrocytes, promoting detoxification in liver and up-regulation of hematopoietic factors in mice. Erythrocyte immune function can clear CIC, promote phagocytosis, present antigens and activate complement, which is mainly achieved by erythrocytes complement receptor type one (CR1). CR1 binds to CIC, viruses, bacteria and tumor cells, and prevents spreading to susceptible tissues, which reflects on E-C3bRR and E-ICRR (Garratty, 2010). EC3bRR represents the quantity and activity of CR1, and E-ICRR represents the content of CIC (Paccaud et al., 1990). In our study, LYC administration significantly increased E-C3bRR and decreased E-ICRR, indicating that LYC ameliorated AFB1-induced erythrocyte immune dysfunction. As CR1 exists on the erythrocyte membrane, erythrocyte membrane damage might reduce the quantity and activity of CR1 (Zhu
Fig. 3. Effects of LYC on erythrocyte SOD and CAT activities in AFB1-exposed mice. (A) SOD; (B) CAT. All data were expressed as means ± SD (n = 6). ⁎⁎p < .01 symbol for the significance of differences versus the CG. ##p < .01 symbol for the significance of differences versus the AG. CG control group, AG AFB1 group, ALG AFB1 + LYC group and LG LYC group. 106
Research in Veterinary Science 129 (2020) 103–108
J. Zhang, et al.
AFB1. LYC also protects lymphocyte against oxidative stress, improving the immune function (Böhm et al., 1995; Yonar, 2012). Thus, we deduced that the protective effects of LYC on erythrocyte immune dysfunction induced by AFB1 are associated with alleviating oxidative stress. However, the limitation of this study is without researching the mechanism of LYC attenuates oxidative stress induced by AFB1, which needs to be elucidated in our further studies. Interestingly, we found that LYC administration significantly increased the PLT level, indicating that LYC may alleviate the coagulation dysfunction induced by AFB1. PLT is the fragments of cytoplasm and derives from the megakaryocytes of bone marrow (Everts et al., 2006). Li et al. found that LYC can protect bone marrow mesenchymal stem cells (MSCs) against ischemiainduced apoptosis in vitro (Li et al., 2014). The patent suggests that MSCs can be used for megakaryocytes production (Cheng et al., 2000). In light of these, we deduced that LYC may protect MSCs and promote megakaryocytes production to alleviate the coagulation dysfunction induced by AFB1. In addition, the effect of LYC on megakaryocytes production and coagulation function exposed AFB1 is worth further studying.
on the ototoxicity induced by cisplatin. Turk J Med Sci 44, 582–585. Costa-Rodrigues, J., Pinho, O., Prr, M., 2018. Can lycopene be considered an effective protection against cardiovascular disease? Food Chem. 245, 1148–1153. Dalel, B., Chayma, B., Yousra, A., 2011. Chemopreventive effect of cactusOpuntia ficus indicaon oxidative stress and genotoxicity of aflatoxin B1. Nutr Metab 8, 73. Everts, P.A., Knape, J.T., Weibrich, G., Schönberger, J.P., Hoffmann, J., Overdevest, E.P., Box, H.A., Van, Z.A., 2006. Platelet-rich plasma and platelet gel: a review. J Extra Corpor Technol 38, 174. Farag, M.R., Alagawany, M., 2017. Erythrocytes as a biological model for screening of xenobiotics toxicity. Chem. Biol. Interact. 279, 73. Freeman, B.A., Crapo, J.D., 1981. Hyperoxia increases oxygen radical production in rat lungs and lung mitochondria. J. Biol. Chem. 256, 10986. Garratty, G., 2010. Advances in red blood cell immunology 1960 to 2009. Transfusion 50, 526–535. Giovannucci, E., 1999. Response: re: tomatoes, tomato-based products, lycopene, and prostate cancer: review of the epidemiologic literature. J. Natl. Cancer Inst. 91, 1331. Giovannucci, E., Rimm, E.Y., Stampfer, M.J., Willett, W.C., 2003. A prospective study of cruciferous vegetables and prostate cancer. Cancer Epidem Biomar 12, 1403–1409. Gong, Y., Hounsa, A., Egal, S., Turner, P.C., Sutcliffe, A.E., Hall, A.J., Cardwell, K., Wild, C.P., 2004. Postweaning exposure to aflatoxin results in impaired child growth: a longitudinal study in Benin, West Africa. Environ Health Persp 112, 1334–1338. Greer, J.P., Arber, D.A., Rodgers, G.M., Paraskevas, F., Foerster, J., Glader, B.E., Means, R.T., 2008. Wintrobe's Clinical Hematology 2 vols 12e. Lippincott Williams & Wilkins. Groopman, J.D., Kensler, T.W., 2005. Role of metabolism and viruses in aflatoxin-induced liver cancer. Toxicol Appl Pharm 206, 131–137. Gupta, N.P., Kumar, R., 2002. Lycopene therapy in idiopathic male infertility—a preliminary report. Int. Urol. Nephrol. 34, 369–372. Herzog, A., Siler, U., Spitzer, V., Seifert, N., Denelavas, A., Hunziker, P.B., Hunziker, W., Goralczyk, R., Wertz, K., 2005. Lycopene reduced gene expression of steroid targets and inflammatory markers in normal rat prostate. FASEB J. 19, 272–274. Joint FAO/WHO Expert Committee on Food Additives, WH Organization, 2000. Evaluation of certain food additives. In: Fifty-First Report of the Joint FAO/WHO Expert Committee on Food Additives. World Health Organization. Kanbur, M., Eraslan, G., Sarica, Z.S., Aslan, O., 2011. The effects of evening primrose oil on lipid peroxidation induced by subacute aflatoxin exposure in mice. Food Chem. Toxicol. 49, 1960–1964. Karahan, I., Ateşşahin, A., Yilmaz, S., Ceribaşi, A.O., Sakin, F., 2005. Protective effect of lycopene on gentamicin-induced oxidative stress and nephrotoxicity in rats. Toxicology 215, 198–204. Kececi, T., Oguz, H., Kurtoglu, V., Demet, O., 1998. Effects of polyvinylpolypyrrolidone,synthetic zeolite and bentonite on serum biochemical and haematological characters of broiler chickens during aflatoxicosis. Br. Poult. Sci. 39, 452–458. Kurt, D., Saruhan, B.G., Yokus, B., Cakir, D.U., 2009. Effects of lycopene and vitamine E administration over gastric mucosal damage induced by aflatoxin B. Journal of Animal & Veterinary Advances 8, 1256–1262. Lanza, G.M., Washburn, K.W., Wyatt, R.D., 1980. Strain variation in hematological response of broilers to dietary aflatoxin. Poultry Sci 59, 2686–2691. Leal, M., Shimada, A., Ruíz, F., González, dM E., 1999. Effect of lycopene on lipid peroxidation and glutathione-dependent enzymes induced by T-2 toxin in vivo. Toxicol. Lett. 109, 1–10. Lewis, L., Onsongo, M., Njapau, H., Schurz-Rogers, H., Luber, G., Kieszak, S., Nyamongo, J., Backer, L., Dahiye, A.M., Misore, A., 2005. Aflatoxin contamination of commercial maize products during an outbreak of acute aflatoxicosis in eastern and central Kenya. Environ. Health Perspect. 113, 1763–1767. Li, W., Jiang, B., Cao, X., Xie, Y., Huang, T., 2016. Protective effect of lycopene on fluoride-induced ameloblasts apoptosis and dental fluorosis through oxidative stressmediated caspase pathways. Chem. Biol. Interact. 261, 27–34. Li, Y., Xue, F., Xu, S.Z., Wang, X.W., Tong, X., Lin, X.J., 2014. Lycopene protects bone marrow mesenchymal stem cells against ischemia-induced apoptosis in vitro. Eur. Rev. Med. Pharmacol. Sci. 18, 1625–1631. Limpens, J., Schröder, F.H., de Ridder, C.M., Bolder, C.A., Wildhagen, M.F., ObermüllerJevic, U.C., Krämer, K., van Weerden, W.M., 2006. Combined lycopene and vitamin E treatment suppresses the growth of PC-346C human prostate cancer cells in nude mice. J. Nutr. 136, 1287. Lin, J., Zhao, H.S., Xiang, L.R., Xia, J., Wang, L.L., Li, X.N., Li, J.L., Zhang, Y., 2016. Lycopene protects against atrazine-induced hepatic ionic homeostasis disturbance by modulating ion-transporting ATPases. J. Nutr. Biochem. 27, 249–256. Majid, G.A., Noosha, Z.J., 2014. Effect of nanosilver on blood parameters in chickens having aflatoxicosis. Toxicol. Ind. Health 30, 192–196. Mein, J.R., Lian, F., Wang, X.D., 2008. Biological activity of lycopene metabolites: implications for cancer prevention. Nutr. Rev. 66, 667–683. Nagababu, E., Chrest, F.J., Rifkind, J.M., 2003. Hydrogen-peroxide-induced heme degradation in red blood cells: the protective roles of catalase and glutathione peroxidase. Biochim. Biophys. Acta 1620, 211–217. Naseer, O., Khan, J.A., Khan, M.S., Omer, M.O., Chishti, G.A., Sohail, M.L., Saleem, M.U., 2016. Comparative efficacy of Silymarin and Choline Chloride (Liver Tonics) in preventing the effects of aflatoxin B1 in bovine calves. Pol. J. Vet. Sci. 19, 545–551. Paccaud, J.P., Carpentier, J.L., Schifferli, J.A., 1990. Difference in the clustering of complement receptor type 1 (CR1) on polymorphonuclear leukocytes and erythrocytes: effect on immune adherence. Eur. J. Immunol. 20, 283–289. Porrini, M., Riso, P., 2000. Lymphocyte lycopene concentration and DNA protection from oxidative damage is increased in women after a short period of tomato consumption. J. Nutr. 130, 189–192. Radhakrishnan, K., Bishop, J., Jin, Z., Kothari, K., Bhatia, M., George, D., Garvin Jr., J.H.,
5. Conclusions In summary, LYC exerts protective effects against AFB1-induced erythrocyte dysfunction and oxidative stress in mice. The findings indicate LYC can be used as a protective agent against AFB1-induced erythrocyte toxicity. Declaration of Competing Interest The authors declare no potential conflicts. Acknowledgments This work was supported by a research grant from the open project program of Northeastern Science Inspection Station, China Ministry of Agriculture Key Laboratory of Animal Pathogen Biology (No.DY201709). References Agarwal, S., Sharma, V., Kaul, T., Abdin, M.Z., Singh, S., 2014. Cytotoxic effect of carotenoid phytonutrient lycopene on P. falciparum infected erythrocytes. Mol. Biochem. Parasitol. 197, 15–20. Amstad, P., Levy, A., Emerit, I., Cerutti, P., 1984. Evidence for membrane-mediated chromosomal damage by aflatoxin B1 in human lymphocytes. Carcinogenesis 5, 719–723. And, D.L.E., Gallagher, E.P., 1994. Mechanisms of aflatoxin carcinogenesis. Annu. Rev. Pharmacol. Toxicol. 34, 135–172. Atessahin, A., Yilmaz, S., Karahan, I., Ceribasi, A.O., Karaoglu, A., 2005. Effects of lycopene against cisplatin-induced nephrotoxicity and oxidative stress in rats. Toxicology 212, 116–123. Bennett, J.W., Klich, M., 2003. Mycotoxins. Clin. Microbiol. Rev. 16, 497–516. Bhuvaneswari, V., Nagini, S., 2005. Lycopene: a review of its potential as an anticancer agent. Curr Med Chem Anticancer Agents 5, 627–635. Boeira, S.P., Filho, C.B., Del’Fabbro, L., Roman, S.S., Royes, L.F.F., Fighera, M.R., Jessé, C.R., Oliveira, M.S., Furian, A.F., 2014. Lycopene treatment prevents hematological, reproductive and histopathological damage induced by acute zearalenone administration in male Swiss mice. Exp. Toxicol. Pathol. 66, 179–185. Böhm, F., Tinkler, J.H., Truscott, T.G., 1995. Carotenoids protect against cell membrane damage by the nitrogen dioxide radical. Nat. Med. 1, 98–99. Boonyaratpalin, M., Supamattaya, K., Verakunpiriya, V., Suprasert, D., 2001. Effects of aflatoxin B1 on growth performance, blood components, immune function and histopathological changes in black tiger shrimp (Penaeus monodon Fabricius). Aquac. Res. 32, 388–398. Cheng, L., Qasba, P., Vanguri, P., Thiede, M.A., 2000. Human mesenchymal stem cells support megakaryocyte and pro-platelet formation from CD34(+) hematopoietic progenitor cells. J. Cell. Physiol. 184, 58–69. Cheung, C.C., Zheng, G.J., Li, A.M., Richardson, B.J., Lam, P.K., 2001. Relationships between tissue concentrations of polycyclic aromatic hydrocarbons and antioxidative responses of marine mussels, Perna viridis. Aquat. Toxicol. 52, 189–203. Chung, W.M., Lin, J.K., Wu, K.C., Hsiung, K.P., 1985. In vitro interconversion of aflatoxin B1 and aflatoxicol by rat erythrocytes. Biochem. Pharmacol. 34, 2566. Ciçek, M.T., Kalcioğlu, T.M., Bayindir, T., Toplu, Y., Iraz, M., 2014. The effect of lycopene
107
Research in Veterinary Science 129 (2020) 103–108
J. Zhang, et al.
aflatoxicosis in rabbits. Bull. Environ. Contam. Toxicol. 49, 861–865. Walker, H.K., Hall, W.D., Hurst, J.W., 1990. Clinical methods:the history, physical, and laboratory examinations. Journal of the American Medical Association 264 (21), 2808–2809. Wang, C.H., Peng, X., Cui, H.M., Fang, J., 2015. Effects of aflatoxin B1 on the erythrocyte count, the content of hemoglobin, and the immune adherence function of erythrocytes in chickens. Med Weter 71, 758–762. Williams, J.H., Phillips, T.D., Jolly, P.E., Stiles, J.K., Jolly, C.M., Aggarwal, D., 2004. Human aflatoxicosis in developing countries: a review of toxicology, exposure, potential health consequences, and interventions. Am. J. Clin. Nutr. 80, 1106–1122. Wintrobe, M.M., 1974. Clinical hematology. South. Med. J. 35, 780–782. Xu, F., Yu, K., Yu, H., Wang, P., Song, M., Xiu, C., Li, Y., 2017. Lycopene relieves AFB 1 -induced liver injury through enhancing hepatic antioxidation and detoxification potential with Nrf2 activation. J. Funct. Foods 39, 215–224. Yener, Z., Celik, I., Ilhan, F., Bal, R., 2009. Effects of Urtica dioica L. seed on lipid peroxidation, antioxidants and liver pathology in aflatoxin-induced tissue injury in rats. Food Chem. Toxicol. 47, 418–424. Yonar, M.E., 2012. The effect of lycopene on oxytetracycline-induced oxidative stress and immunosuppression in rainbow trout (Oncorhynchus mykiss, W.). Fish Shellfish Immun 32, 994–1001. Yonar, S.M., 2013. Toxic effects of malathion in carp, Cyprinus carpio carpio: protective role of lycopene. Ecotox Environ Safe 97, 223–229. Zhao, Y.Z., Jia, J., Li, Y.B., Guo, C.X., Zhou, X.Q., Sun, Z.W., 2014. Effects of endosulfan on the immune function of erythrocytes, and potential protection by testosterone propionate. J. Toxicol. Sci. 39, 701–710. Zhu, Y., Zhao, H., Li, X., Zhang, L., Hu, C., Shao, B., Sun, H., Bah, A.A., Li, Y., Zhang, Z., 2011. Effects of subchronic aluminum exposure on the immune function of erythrocytes in rats. Biol. Trace Elem. Res. 143, 1576–1580.
Martinez, M., Ovchinsky, N., Lobritto, S., Elsayed, Y., Satwani, P., 2013. Risk factors associated with liver injury and impact of liver injury on transplantation-related mortality in pediatric recipients of allogeneic hematopoietic stem cell transplantation. Biol Blood Marrow Transplant 19, 912–917. Rothschild, L.J., 1992. IARC classes AFB1 as class 1 human carcinogen. Food Chem. News 34, 62–66. Schaer, D.J., Buehler, P.W., Alayash, A.I., Belcher, J.D., Vercellotti, G.M., 2013. Hemolysis and free hemoglobin revisited: exploring hemoglobin and hemin scavengers as a novel class of therapeutic proteins. Blood 121, 1276–1284. Siegel, I., Liu, T.L., Gleicher, N., 1981. The red-cell immune system. Lancet 2, 556. Smith, L.E., Prendergast, A.J., Turner, P.C., Humphrey, J.H., Stoltzfus, R.J., 2017. Aflatoxin exposure during pregnancy, maternal anemia, and adverse birth outcomes. Am J Trop Med 96, 770–776. Stahl, W., Sies, H., 2003. Antioxidant activity of carotenoids. Mol. Asp. Med. 24, 345–351. Tang, L., Guan, H., Ding, X., 2007. Modulation of aflatoxin toxicity and biomarkers by lycopene in F344 rats. Toxicol Appl Pharm 219, 10–17. Tennant, B., Harrold, D., Reina-Guerra, M., Kendrick, J.W., Laben, R.C., 1974. Hematology of the neonatal calf: erythrocyte and leukocyte values of normal calves. Cornell Veterinarian 64, 516. Toghyani, M., Tohidi, M., Gheisari, A.A., Tabeidian, S.A., 2010. Performance, immunity, serum biochemical and hematological parameters in broiler chicks fed dietary thyme as alternative for an antibiotic growth promoter. Afr. J. Biotechnol. 9, 6819–6825. Ural, M.Ş., 2013. Chlorpyrifos-induced changes in oxidant/antioxidant status and haematological parameters of Cyprinus carpio carpio: ameliorative effect of lycopene. Chemosphere 90, 2059–2064. Verma, R.J., 2004. Aflatoxin cause DNA damage. Int. J. Hum. Genet. 4 (4), 231–236. Verma, R.J., Raval, P.J., 1991. Cytotoxicity of aflatoxin on red blood corpuscles. Bull. Environ. Contam. Toxicol. 47, 428–432. Verma, R.J., Raval, P.J., 1992. Alterations in erythrocytes during induced chronic
108