Saffron (Crocus Sativus Linnaeus) based protection against Aflatoxin B1 induced organ damage in rats

Hayat Ashi; Enas Hamed; Bassem  Refaat; Shakir Idris; Latifa Khayyat; Tasahil Albishi; Leena Neyaz; Outour Alami; Fatimah Al-Rahmani; Shirin Aashi; Abdulaziz Alamri; Ghazi Abduljawad; Ayman Alobaidi; Fahad Alburberry; Saleh Alsalhi; Rayan Wali; Khaled Elbanna; Hussein Abulreesh

doi:10.14720/abs.67.3.18966

Authors

Hayat Ashi Department of Biology, Faculty of Science, Umm Al-Qura University, Makkah 21955, Saudi Arabia
Enas Hamed Department of Medical Physiology, Faculty of Medicine, Assiut University, Assiut, Egypt
Bassem Refaat Department of Clinical Laboratory Sciences, Faculty of Applied Medical Sciences, Umm Al-Qura University, Makkah 21955, Saudi Arabia
Shakir Idris Department of Clinical Laboratory Sciences, Faculty of Applied Medical Sciences, Umm Al-Qura University, Makkah 21955, Saudi Arabia
Latifa Khayyat Department of Biology, Faculty of Science, Umm Al-Qura University, Makkah 21955, Saudi Arabia
Tasahil Albishi Department of Biology, Faculty of Science, Umm Al-Qura University, Makkah 21955, Saudi Arabia
Leena Neyaz Department of Biology, Faculty of Science, Umm Al-Qura University, Makkah 21955, Saudi Arabia
Outour Alami Department of Biology, Faculty of Science, Umm Al-Qura University, Makkah 21955, Saudi Arabia
Fatimah Al-Rahmani Department of Chemistry, Faculty of Science, Umm Al-Qura University, Makkah 21955, Saudi Arabia
Shirin Aashi College of Pharmacy, King Abdulaziz University, Jeddah, Saudi Arabia
Abdulaziz Alamri College of Pharmacy, King Abdulaziz University, Jeddah, Saudi Arabia
Ghazi Abduljawad College of Pharmacy, King Abdulaziz University, Jeddah, Saudi Arabia
Ayman Alobaidi College of Pharmacy, King Abdulaziz University, Jeddah, Saudi Arabia
Fahad Alburberry College of Pharmacy, King Abdulaziz University, Jeddah, Saudi Arabia
Saleh Alsalhi College of Pharmacy, King Abdulaziz University, Jeddah, Saudi Arabia
Rayan Wali College of Pharmacy, King Abdulaziz University, Jeddah, Saudi Arabia
Khaled Elbanna Department of Biology, Faculty of Science, Umm Al-Qura University, Makkah 21955, Saudi Arabia
Hussein Abulreesh Umm Al-Qura University

DOI:

https://doi.org/10.14720/abs.67.3.18966

Keywords:

Aflatoxin B1, antioxidant, Hemotoxicity, Hepatotoxicity, Saffron, oxidative stress

Abstract

Saffron is well-known for its anti-apoptotic, anti-inflammatory, and antioxidant properties. Saffron's nutritional and medicinal properties support its numerous uses as a flavouring and herbal remedy. This study investigated the protective efficacy of saffron administration against aflatoxin B1 (AFB1)-induced toxicity in adult male Wistar albino rats during an experimental period of 21 days. Aflatoxin B1 (AFB1) is a common mycotoxin of soils and foodstuffs. Thirty-two rats were divided into four groups (Control group, AFB1 group, Saffron group, and AFB1+ Saffron group), and their body weights were measured on days 1, 7, 14, and 21. Blood samples were collected on the 21st day for haematological and biochemical studies (testosterone, kidney and liver function tests, and oxidative stress markers). Tissue samples from testes, liver, and kidney were subjected to histological examinations. The results depicted a significant decrease in the body weights after 7, 14, and 21 days of Saffron, AFB1, and AFB1+ Saffron treatments in comparison to control. Haematological investigations showed that basophils, platelets, monocytes, lymphocytes, and eosinophils greatly increased compared to the control group, whereas neutrophils and eosinophils dramatically decreased. There was a significant rise in the serum levels of uric acid, creatinine, aspartate transaminase, alkaline phosphatase, nitric oxide, and malondialdehyde. Contrarily, testosterone levels notably reduced in AFB1-administered rats as compared to controls. AFB1 group exhibited several histological modifications in testes, liver, and kidney tissues. Oxidative stress biomarkers, testosterone, kidney and liver functions, and haematological parameters of the AFB1+ Saffron group remained similar to the control group. Kidney and liver tissues of Saffron-treated rats also displayed normal structure similar to the control group, which confirmed its protective efficacy against AFB1-induced toxicity. Saffron's bioactive components and antioxidant and pharmacological properties might have contributed to its promising anti-AFB1-toxicity potential.

Metrics

Metrics Loading ...

References

Abdel-Aziem, S.H., Hassan, A.M., Abdel-Wahhab, M.A., 2011. Dietary supplementation with whey protein and ginseng extract counteracts oxidative stress and DNA damage in rats fed an aflatoxin-contaminated diet. Mutation Research/Genetic Toxicology and Environmental Mutagenesis, 723, 65-71. DOI:10.1016/j.mmrgentox.2011.04.007.

Abu El-Saad, A.S., Mahmoud, H.M., 2009. Phytic acid exposure alters aflatoxinB1-induced reproductive and oxidative toxicity in albino rats (Rattus norvegicus). Evidence-Based Complementary and Alternative Medicine, 6, 331-341. DOI:10.1093/ecam/nem173.

Adedara, I.A., Nanjappa, M.K., Farombi, E.O., Akingbemi, B.T., 2014. Aflatoxin B1 disrupts the androgen biosynthetic pathway in rat Leydig cells. Food and Chemical Toxicology 65, 252-9. DOI:10.1016/j.fct.2013.12.027.

Ahamad, D.B., Sharma, A., Dwivedi, P., 2015. Aflatoxin B1 induced carcinogenicity in Wistar rats: Clinical signs and growth performance. Shanlax International Journal of Veterinary Science, 3, 2321-6387.

Akhoundzadeh, B.A., Ghoreyshi, S., Nourbala, A., Akhoundzadeh, S., Rezazadeh, S.(2008).Petal and stigma of Crocus sativus L.in the treatment of depression: a pilot double-blind randomized trial. Progress in Neuro-Psychopharmacology and Biological Psychiatry 31, 439-442. DOI:10.1016/j.pnpbp.2006.11.010.

Ali, A., Khatoon, A., Almohaimeed, H.M., Al-Sarraj, F., Albiheyri, R., Alotibi, I., Abidin, Z.U., 2022. Mitigative potential of novel Lactobacillus plantarum TISTR 2076 against the aflatoxins-associated oxidative stress and histopathological alterations in liver and kidney of broiler chicks during the entire growth period. Toxins, 14, 689. DOI:10.3390/toxin14100689.

Altinoz, E., Oner, Z., Elbe, H., Cigremis, Y., Turkoz, Y., 2015. Protective effects of saffron (its active constituent, crocin) on nephropathy in streptozotocin-induced diabetic rats. Human, Experimental Toxicology, 34, 127-134. DOI:10.1177/0960327114538989.

Asadi, M.H., Zafari, F., Sarveazad, A., Abbasi, M., Safa, M., Koruji, M., Yari, A., Alizadeh Miran, R., 2014. Saffron improves epididymal sperm parameters in rats exposed to cadmium. Nephro-Urology Monthly 6, e12125. DOI:10.5812/numonthly.12125.

Ashi, H., Almalki, M., Hamed, E.A., Makhlouf, M.M.M., Alsulami, F.S., Alharbi, A.S., Alharbi, A.F., Alami, O.T., Alqethami, N., Al-Rahmani, F., Thabit, M.A., Aashi, S., Alzahrani, Y.A., Elbanna, K., Abulreesh, H.H., 2023a. Effect of aflatoxin B1 on the haematology, testes, and lungs of adult male rats: a physiologal and histological study. Animal Science and Genetics 19, 93-114. DOI:https://doi.org/10.5604/01.3001.0053.9337.

Ashi, H., Almalki, M.H., Hamed, E.A., Ramadan, W.S., Alahmadi, T.F., Alami, O.T., Arafa, S.H., Alshareef, A.K., Alsulami, F.S., Alharbi, A.F., Al-Harbi, M.S., Alqurashi, E.H., Aashi, S., Alzahrani, Y.A., Elbanna, K., Abulreesh, H.H., 2023b.Protective and therapeutic effects of lactic acid bacteria against aflatoxin B1 toxicity to rat organs. Microorganisms 11, 1703. DOI:https;//doi.org/10.3390/microorganisms11071703.

Babaei, A., Arshami, J., Haghparast, A., Mesgaran, M.D., 2014. Effects of saffron (Crocus sativus) petal ethanolic extract on hematology, antibody response, and spleen histology in rats. Avicenna journal of phytomedicine, 4, 103.

Bertrand, S., Schumpp, O., Bohni, N., Bujard, A., Azzollini, A., Monod, M., Gindro, K., Wolfender, J.L., 2013. Detection of metabolite induction in fungal co-cultures on solid media by high-throughput differential ultra-high pressure liquid chromatography-time-of-flight mass spectrometry fingerprinting. Journal of Chromatography A, 1292, 219-28. DOI:10.1016/j.chroma.2013.01.098.

Bolhassani, A., 2018. Bioactive components of saffron and their pharmacological properties. Studies in Natural Products Chemistry, 58, 289-311. DOI:10.1016/B978-0-444-640056-7.00010-6.

Cao, Z., Shao, B., Xu, F., Liu, Y., Li, Y., Zhu, Y., 2017. Protective effect of selenium on aflatoxin b1-induced testicular toxicity in mice. Biological Trace Elemement Research 180, 233-238. DOI:10.1007/s12011-017-0997-z.

Dai, Y., Huang, K., Zhang, B., Zhu, L., Xu, W., 2017. Aflatoxin B1-induced epigenetic alterations: An overview. Food and Chemical Toxicology 109, 683-689. DOI:10.1016/j.fct.2017.06.034.

Daryoush, M., Yousef, D., 2012. Protective effect of ethanolic extracte of Crocus sativus L.[Saffron] stigma against Cisplatin induced hepatotoxicity in rats. Medical Sciences 21, 251-261.

Esmaeili, N., Ebrahimzadeh, H., Abdi, K., Safarian, S., 2011. Determination of some phenolic compounds in Crocus sativus L. corms and its antioxidant activities study. Pharmacognosy Magazine, 7, 74. DOI:10.4103/0973-1296.75906.

Faisal, K., Periasamy, V., Sahabudeen, S., Radha, A., Anandhi, R., Akbarsha, M., 2008. Spermatotoxic effect of aflatoxin B1 in rat: extrusion of outer dense fibres and associated axonemal microtubule doublets of sperm flagellum. Reproduction, 135, 303-310. DOI:10.1530/REP--07-0367.

Fakruddin, M., Chowdhury, A., Hossain, M.N., Ahmed, M.M., 2015. Characterization of aflatoxin producing Aspergillus flavus from food and feed samples. Springerplus, 4, 159. DOI:10.1186/s40064-015-097-1.

Giaccio, M., 2004. Crocetin from saffron: an active component of an ancient spice. Critical Reviews in Food Science and Nutrition, 44, 155-172. DOI:10.1080/10408690490441433.

Harchegani, A.B., Abolfazl, K., Mahdiyeh Mirnam, N., Kaboutaraki, H.B., Shirvani, H., Shahriary, A., 2019. The hepatoprotective and antioxidative effect of saffron stigma alcoholic extract against vincristine sulfate induced toxicity in rats. Interdisciplinary Toxicology, 12, 186. DOI:10.2478/intox-2019-0023.

Hariri, A.T., Moallem, S.A., Mahmoudi, M., Memar, B., Hosseinzadeh, H., 2010. Sub-acute effects of diazinon on biochemical indices and specific biomarkers in rats: protective effects of crocin and safranal. Food and Chemical Toxicology, 48, 2803-2808. DOI:10.1016/j.fct.2010.07.010.

Heidary, M., Vahhabi, S., Reza Nejadi, J., Delfan, B., Birjandi, M., Kaviani, H., Givrad, S., 2008. Effect of saffron on semen parameters of infertile men.Urology Journal 5, 255-9.

Hosseinzadeh, H., Shariaty, V.M., 2007. Anti-nociceptive effect of safranal, a constituent of Crocus sativus (saffron), in mice. Pharmacologyonline, 2, 498-503.

Https://Www.Istockphoto.Com/Vector/Structure-of-Aspergillus-Gm1222027247-358441161.[Accessed 20 Feb., 2024].

Husain, A.S., Thalij, K.M., Dheeb, B.I., 2014. Effects of interaction between Aflatoxins (AFs) and functional materials FM in the hematological, biochemical parameters and enzyme activity in Rats. Egyptian Academic Journal of Biological Sciences, B. Zoology, 6, 17-22.

Iranshahi, M., Khoshangosht, M., Mohammadkhani, Z., Karimi, G., 2011. Protective effects of aqueous and ethanolic extracts of saffron stigma and petal on liver toxicity induced by carbon tetrachloride in mice. Pharmacologyonline, 1, 203-212.

Karaca, A., Yilmaz, S., Kaya, E., Altun, S., 2021. The effect of lycopene on hepatotoxicity of aflatoxin B1 in rats. Archives of Physiology and Biochemistry, 127, 429-436. DOI:10.1080/13813455.2019.1648516.

Karmanov, A.P., Kanarsky, A.V., Kocheva, L.S., Semenov, E.I., Belyy, V.A., 2021. In vitro study of adsorption efficiency of natural lignins towards aflatoxin B2. Reactive and Functional Polymers, 167, 105033. DOI:10.1016/j.reactfunctpolym.2021.105033.

Khaled, M.Q., Thalij, K.M., 2021. Effect of aflatoxin B1 contaminated corn and their products on some physiology parameters in laboratory rats. IOP Conference Series: Earth and Environmental Science, IOP Publishing, 012103.

Kheir Eldin, A.A., Motawi, T.M., Sadik, N.A., 2008. Effect of some natural antioxidants on aflatoxin B1-induced hepatic toxicity. EXCLI Journal, 7, 119-131.

Koul, A., Abraham, S.K., 2017. Intake of saffron reduces γ-radiation-induced genotoxicity and oxidative stress in mice. Toxicology Mechanisms and Methods, 27, 428-434. DOI:10.1080/15375616.2017.1307476.

Kudayer, A.M., Alsandaqchi, A.T., Saleh, F.M., Alwan, N.A., 2019. Toxic effect of aflatoxin B1 on heart, lung, and testis of male albino rats: histopathology study. IOP Conf.Series: Materials Science and Engineering, 571, 012055.

Lahmass, I., El Khoudri, M., Ouahhoud, S., Lahmass, M., Khoulati, A., Benyoussef, S., Mamri, S., Meziane, M., Saalaoui, E., 2021. Biological effects and pharmacological activities of saffron of Crocus sativus. Arabian Journal of Medicinal and Aromatic Plants, 7, 254-68.

Li, M., Kong, Y., Guo, W., Wu, X., Zhang, J., Lai, Y., Kong, Y., Niu, X., Wang, G., 2022. Dietary aflatoxin B1 caused the growth inhibition, and activated oxidative stress and endoplasmic reticulum stress pathway, inducing apoptosis and inflammation in the liver of northern snakehead (Channa argus). Science of The Total Environment, 850, 157997. DOI:10.1016/j.scitotenv.2022.157997.

Li, P., Zhang, Q., Zhang, D., Guan, D., Liu, D.X., Fang, S., Wang, X., Zhang, W., 2011. Aflatoxin measurement and analysis. In: Torres-Pacheco I.(Ed.), Aflatoxins-Detection, Measurement and Control.Pp.183-208, IntechOpen, Croatia.

Modaresi, M., Messripour, M, Asadi, M., Marghmaleki,, Hamadanian, M.K., 2008. Effect of saffron [Crocus sativus] extract on level of FSH, LH and testosterone in mice. Journal of Zanjan University of Medical Sciences and Health Services, 16, 11-17.

Mahmoudzadeh, L., Najafi, H., Ashtiyani, S.C., Yarijani, Z.M., 2017. Anti‐inflammatory and protective effects of saffron extract in ischaemia/reperfusion‐induced acute kidney injury. Nephrology, 22, 748-754.

Manchón, N., D’arrigo, M., García-Lafuente, A., Guillamón, E., Villares, A., Ramos, A., Martínez, J., Rostagno, M., 2010. Fast analysis of isoflavones by high-performance liquid chromatography using a column packed with fused-core particles. Talanta, 82, 1986-1994.

Mashmoul, M., Azlan, A., Mohtarrudin, N., Mohd Yusof, B.N., Khaza'ai, H., Khoo, H.E., Farzadnia, M., Boroushaki, M.T., 2016. Protective effects of saffron extract and crocin supplementation on fatty liver tissue of high-fat diet-induced obese rats.BMC Complementary and Alternative Medicine, 16, 401. DOI:10.1186/s112906-016-1381-9.

Mirhadi, E., Nassirli, H., Malaekeh-Nikouei, B., 2020. An updated review on therapeutic effects of nanoparticle-based formulations of saffron components (safranal, crocin, and crocetin). Journal of Pharmaceutical Investigation, 50, 47-58.

Moallem, S.A., Hariri, A.T., Mahmoudi, M., Hosseinzadeh, H., 2014. Effect of aqueous extract of Crocus sativus L.(saffron) stigma against subacute effect of diazinon on specific biomarkers in rats. Toxicology and Industrial Health, 30, 141-146. DOI:10.1177/0748233712452609.

Mtimet, N., Walke, M., Baker, D., Lindahl, J., Hartmann, M., Grace, D., 2015. Kenyan awareness of aflatoxin: An analysis of processed milk consumers. International Conference of Agricultural Economists, Milan, Italy.

Nielsen, K.F., Smedsgaard, J., 2003. Fungal metabolite screening: database of 474 mycotoxins and fungal metabolites for dereplication by standardised liquid chromatography-UV-mass spectrometry methodology. Journal of Chromatography A, 1002, 111-36.

Noorbala, A., Akhondzadeh, S., Tahmacebi-Pour, N., Jamshidi, A., 2005. Hydro-alcoholic extract of Crocus sativus L.versus fluoxetine in the treatment of mild to moderate depression: a double-blind, randomized pilot trial. Journal of Ethnopharmacology, 97, 281-284.

Omidi, A., Riahinia, N., Torbati, M.B.M., Behdani, M.-A., 2014. Hepatoprotective effect of Crocus sativus (saffron) petals extract against acetaminophen toxicity in male Wistar rats. Avicenna Journal of Phytomedicine, 4, 330.

Owumi, S.E., Danso, O.F., Effiong, M.E., 2019. Dietary quercetin abrogates hepatorenal oxidative damage associated with dichloromethane exposure in rats. Acta Biochimica Polonica, 66, 201-206. DOI:10.18388/abp.2018_2771.

Pan, T.L., Wu, T.H., Wang, P.W., Leu, Y.L., Sintupisut, N., Huang, C.H., Chang, F.R., Wu, Y.C., 2013. Functional proteomics reveals the protective effects of saffron ethanolic extract on hepatic ischemia‐reperfusion injury. Proteomics, 13, 2297-2311.

Pandita, D., 2021. Saffron (Crocus sativus L.): Phytochemistry, therapeutic significance and omics-based biology. In: Aftab T., Hakeem K.R.(Eds.), Medicinal and Aromatic Plants. Pp.325-369. Academic Press, USA. DOI:10.1016/B978-0-12-819590-1.00014-8.

Parasuraman, S., Raveendran, R., Kesavan, R., 2010. Blood sample collection in small laboratory animals. Journal of Pharmacology, Pharmacotheraputics, 1, 87-93. DOI:10.4103/0976-500X.72350.

Popescu, R.G., Rădulescu, A.L., Georgescu, S.E., Dinischiotu, A., 2022. Aflatoxins in feed: types, metabolism, health consequences in swine and mitigation strategies. Toxins, 14, 853. DOI:10.3390/toxins14120853.

Ramamurthy, V., Rajakumar, R., 2016. Studies on ethanolic leaf extract of phyllanthus niruri and its effect on aflatoxin intoxicated male albino rats. International Journal of Zoology and Applied Biosciences, 1, 1-6.

Rezaee, R., Hosseinzadeh, H., 2013. Safranal: from an aromatic natural product to a rewarding pharmacological agent. Iranian Journal of Basic Medical Sciences, 16, 12.

Rotimi, O.A., Rotimi, S.O., Goodrich, J.M., Adelani, I.B., Agbonihale, E., Talabi, G., 2019. Time-course effects of acute aflatoxin B1 exposure on hepatic mitochondrial lipids and oxidative stress in rats. Frontiers in Pharmacology, 10, 467. DOI:10.3389/fphar.2019.00467.

Rushing, B.R., Selim, M.I., 2019. Aflatoxin B1: A review on metabolism, toxicity, occurrence in food, occupational exposure, and detoxification methods. Food and Chemical Toxicology, 124, 81-100. DOI:10.1016/j.fct.2018.11.047.

Saito, M., Machida, S., 1999. A rapid identification method for aflatoxin-producing strains of Aspergillus flavus and A.parasiticus by ammonia vapor. Mycoscience, 40, 205-208.

Sakr, S.A., Zowail, M.E., Marzouk, A.M., 2014. Effect of saffron (Crocus sativus L.) on sodium valporate induced cytogenetic and testicular alterations in albino rats. Anatomy and Cell Biology, 47, 171-9. DOI:10.5115/acb.2014.47.3.171.

Saldan, N.C., Almeida, R.T.R., Avíncola, A., Porto, C., Galuch, M.B., Magon, T.F.S., Pilau, E.J., Svidzinski, T.I.E., Oliveira, C.C., 2018. Development of an analytical method for identification of Aspergillus flavus based on chemical markers using HPLC-MS. Food Chemistry, 241, 113-121. DOI:10.1016/j.foodchem.2017.08.065.

Shabeer, S., Asad, S., Jamal, A., Ali A., 2022. Aflatoxin contamination, its impact and management strategies: an update review. Toxins, 14, 307. DOI:10.3390/toxins14050307.

Shati, A.A., Alamri, S.A., 2010. Role of saffron (Crocus sativus L.) and honey syrup on aluminum-induced hepatotoxicity. Saudi Medical Journal, 31, 1106-1113.

Sreekanth, C.N., Bava, S.V., Sreekumar, E., Anto, R.J., 2011. Molecular evidences for the chemosensitizing efficacy of liposomal curcumin in paclitaxel chemotherapy in mouse models of cervical cancer. Oncogene, 30, 3139-52. DOI:10.1038/onc.2011.23. DOI:10.1016/B978-0-12-818462-2.0001-2.

Supriya, C., Girish, B., Reddy, P.S., 2014. Aflatoxin B1-induced reproductive toxicity in male rats: possible mechanism of action. International Journal of Toxicology, 33, 155-161.

Thomson, M., Bou‐Abbas, F., Alansary, A., Al‐Qattan, K.K., Ali, M., 2009. Effect of saffron on levels of blood lipids in rats fed a high cholesterol diet. The FASEB Journal, 23, 901.4-901.4.

Towner, R.A., Mason, R.P., Reinke, L.A., 2002. In vivo detection of aflatoxin-induced lipid free radicals in rat bile. Biochimca et Biophysica Acta, 1573, 55-62.

Türk, G., Sönmez, M., Aydin, M., Yüce, A., Gür, S., Yüksel, M., Aksu, E.H., Aksoy, H., 2008. Effects of pomegranate juice consumption on sperm quality, spermatogenic cell density, antioxidant activity and testosterone level in male rats. Clinical Nutrition, 27, 289-96. DOI:10.1016/j.clnu.2007.12.006.

Verma, R., Nair, A., 2002. Effect of aflatoxins on testicular steroidogenesis and amelioration by vitamin E. Food and Chemical Toxicology, 40, 669-672.

Wang, D., Lindemann, M.D., Estienne, M.J.,2020. Effect of folic acid supplementation and dietary protein level on growth performance, serum chemistry and immune response in weanling piglets fed differing concentrations of aflatoxin. Toxins, 12, 651. DOI:10.3390/toxins12100651.

Yarijani, Z.M., Pourmotabbed, A., Pourmotabbed, T., Najafi, H., 2017. Crocin has anti-inflammatory and protective effects in ischemia-reperfusion induced renal injuries. Iranian Journal of Basic Medical Sciences, 20, 753.

Yilmaz, S., Kaya, E., Karaca, A., Karatas, O., 2018. Aflatoxin B1 induced renal and cardiac damage in rats: protective effect of lycopene. Research in Veterinary Science, 119, 268-275.

Yuan, G., Dai, S., Yin, Z., Lu, H., Jia, R., Xu, J., Song, X., Li, L., Shu, Y., Zhao, X., 2014. Toxicological assessment of combined lead and cadmium: acute and sub-chronic toxicity study in rats. Food and Chemical Toxicology, 65, 260-268. DOI:10.106/j.fct.2013.12.041.