Muhammad Salihu IBRAHİM, Meltem ÇAKMAK, Dursun ÖZER, Fikret KARATAŞ, Sinan SAYDAM

Kadmiyum ve C Vitamininin Citrobacter Freundii'nin Antioksidan Enzimleri ve Stres Belirteçleri Üzerine Etkisi

Bu çalışmada Citrobacter freundii (NRRL B-2643) değişik konsantrasyonlarda kadmiyum içeren LB besi yerinde üretildi. Kadmiyumun (Cd) oluşturduğu olumsuz etkiyi azaltmak için, kadmiyum içeren besi yerine antioksidan özelliği ile bilinen değişik konsantrasyonlarda C vitamini katılarak da bakteri çoğaltıldı. Çoğaltılan bakteri konsantrasyonu, protein miktarı ve antioksidan enzimlerin (Glutatyon peroksidaz (GSH-Px), Glutatyon redüktaz (GSH-Rd), Süperoksit dismutaz (SOD), Katalaz (CAT) ve peroksidaz (POD)) aktiviteleri spektofotometre ile belirlendi. Ayrıca redükte ve okside glutatyon (GSH ve GSSG), 4-hidroksineoneal (4-HNE) ve malondialdehit (MDA) miktarları ise HPLC ile tayin edildi. 40 ppm kadmiyum konsantrasyonuna kadar bakteri üretiminin etkilenmediği 150 ppm ve daha yüksek Cd konsantrasyonlarında de ise anlamlı mikroorganizma üremesi gözlenemedi. Bu nedenle 0, 75, 100 ve 125 ppm Cd içeren besi yerlerinde bakteri üretimi gerçekleştirildi. 75, 100 ve 125 ppm Cd içeren besi yerinde üretilen mikroorganizmaların protein miktarı kontrole göre sırasıyla yüzde 24, 44 ve 62 oranında azalmıştır. Aynı besi ortamına 50 ppm C vitamini eklendiğinde ise kontrole göre protein miktarındaki yüzde azalma sırasıyla 10, 31 ve 50 olarak bulunmuştur. Kadmiyum içeren besi ortamında üretilen bakterilerdeki antioksidan enzimlerin aktiviteleri ve stres biyomarkerlerı kontrole göre artış, göstermiştir (p<0.05). Kadmiyum içeren besi ortamına 25, 50 ve 75 ppm C vitamini katılmasıyla birlikte antioksidan enzimlerin aktiviteleri ve stres belirteçlerinin miktarlarında azalma olduğu gözlenmiştir.

Anahtar Kelimeler:

Antioksidan Enzimler, Citrobacter freundii, Kadmiyum, Sitres Belirteçleri

Effect of Cadmium and Vitamin C on Citrobacter Freundii's Antioxidant Enzymes and Stress Markers

In this study, Citrobacter freundii (NRRL B-2643) bacteria were grown in LB medium containing varying concentrations of cadmium (Cd). In order to reduce the negative effect of Cd, different concentrations of vitamin C, known for its antioxidant properties, were added to the Cd-containing growth medium. Bacterial concentration, soluble protein and activities of antioxidant enzymes (Glutathione peroxidase (GSH-Px), Glutathione reductase (GSH-Rd), Superoxide dismutase (SOD), Catalase (CAT) and peroxidase (POD)) were determined by spectrophotometer. In addition, reduced and oxidized glutathione (GSH and GSSG), 4-hydroxyneoneal (4-HNE), malondialdehyde (MDA) amounts were determined by HPLC. No significant microorganism growth was observed at 150 ppm and higher Cd concentrations. Bacteria production was not affected up to 40 ppm Cd concentration. Bacteria were grown in media containing 0, 75, 100 and 125 ppm Cd. The protein content of the microorganism grown in the medium containing 75, 100 and 125 ppm Cd decreased about 24, 44 and 62 percent, respectively, comparisons to the control. When 50 ppm of vitamin C was added to the same growth medium, the percentage decrease in protein amount compared to the control was found to be 10, 31 and 50, respectively. An increase was observed in the antioxidant enzymes activities and stress markers in bacteria grown in cadmium-containing media compared to the control (p<0.05). With the addition of 25, 50 and 75 ppm vitamin C to cadmium-containing media, a decrease was observed in the activities of antioxidant enzymes and the amounts of stress markers.

Keywords:

Antioxidant Enzymes, Citrobacter freundii, Cadmium, Stress Markers,

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Adwas, A.A., Elsayed, A.S.I., Azab, A.E. and Quwaydir, F.A., 2019. Oxidative stress and antioxidant mechanisms in human body. Journal of Applied Biotechnology & Bioengineering, 6, 43-47.
Aebi H. 1984. Catalse in vitro. Academy Press, Methods Enzymol. New York, 105, 121-126.
Al-Ghafari, Ayat; Elmorsy, Ekramy; Fikry, Emad; Alrowaili, Majed; Carter, Wayne G.; Mukhopadhyay, Partha, 2019. The heavy metals lead and cadmium are cytotoxic to human bone osteoblasts via induction of redox stress, PLOS ONE, 14(11), e0225341–
Banerjee, G., Pandey, S., Ray, A.K., and Kumar, R., 2015. Bioremediation of heavy metals by a novel bacterial strain Enterobacter cloacae and its antioxidant enzyme activity, flocculant production, and protein expression in presence of lead, cadmium, and nickel. Water, Air, & Soil Pollution, 226, 1-9.
Banfalvi, G., 2011. Cellular Effects of Heavy Metals. Netherlands, London, New York: Springer. ISBN 978-94-007-0428-2.
Beutler, E., 1984. Red cell metabolism. A manual of biochemical methods. 3th Ed. Grune & Stratton Orlando,72-73, 74-75, 105-106. ed, USA.
Beyersmann, D. and Hartwig, A., 2008. Carcinogenic metal compounds: recent insight into molecular and cellular mechanisms. Archives of toxicology, 82, 493-51.
Cheng, J., Qiu, H., Chang, Z., Jiang, Z., and Yin, W., 2016. The effect of cadmium on the growth and antioxidant response for freshwater algae Chlorella vulgaris. SpringerPlus, 5, 1-8.
Choudhary, M., Jetley, U.K., Khan, M.A., Zutshi, S. and Fatma, T., 2007. Effect of heavy metal stress on proline, malondialdehyde, and superoxide dismutase activity in the cyanobacterium Spirulina platensis-S5. Ecotoxicology and environmental safety, 66, 204-209.
Cnubben, N.H.P., Rıetjens, I.M.C.M., Wortelboer, H., Van Zanden, J. and Van Bladeren, P.J., 2001. The interplay of glutathione-related processes in antioxidant defense. Environmental Toxicology and Pharmacology, 10, 141-152.
Corticeiro, S.C., Lima, A.I.G., and Figueira, E.M.d.A.P., 2006. The importance of glutathione in oxidative status of Rhizobium leguminosarum biovar viciae under Cd exposure. Enzyme and microbial technology, 40, 132-137.
Franklin, R., Mark, W., Geraldine, L., Christina, K., Ruchong, O., Lesley, B. and Judy, d.H., (2013). Oxidative stress in surgery in an ageing population, Pathophysiology and therapy. Experimental Gerontology, 48, 45-54.
Gaweł, S., Wardas, M., Niedworok, E. and Wardas, P., 2004. Malondialdehyde as lipid peroxidation marker. Wiadomosci Lekarskie, 57(9-10), 453-455.
Güner, U., 2010. Heavy metal effects on P, Ca, Mg, and total protein contents in embryonic pleopodal eggs and stage-1 juveniles of freshwater crayfish Astacus leptodactylus (Eschscholtz, 1823). Turkish Journal of Biology, 34, 405-412.
Hamza, Amal H., 2017. Vitamin C || Vitamin C: Sources, Functions, Sensing and Analysis, 10.5772/66058 (Chapter 1), DOI:10.5772/intechopen. 7016.
Hussein, K.A. and Joo, J.H., 2013. Heavy metal resistance of bacteria and its impact on the production of antioxidant enzymes. African Journal of Microbiology Research, 7, 2288- 2296.
Ibrahim, M., Ibrahim, Y., Mukhtar, Z. and Karatas F. 2017. Amount of vitamin A, vitamin E, vitamin C, malondialdehyde, glutathione, ghrelin, beta-carotene, lycopene in fruits of Hawthorn, Midland (Crataegus laevigata). Journal of Human Nutrition & Food Science, 5, 1112-1117.
Jemai, H., Messaoudi, I., Chaouch, A. and Kerkeni, A., 2007. Protective Effect of Zinc Supplementation on Blood Antioxidant Defense System in Rats Exposed to Cadmium. Journal of Trace Elements in Medicine and Biology, 21(4), 269–73.
Karatas, F., Karatepe, M. and Baysar, A., 2002. Determination of free malondialdehyde in human serum by high-performance liquid chromatography. Analytical Biochemistry, 311, 76-79.
Kireçci, O.A., 2017. Saccharomyces cerevisiae’nın Gelişme Ortamına İlave Edilen Ağır Metallerin (Mn, Mg, Cd, Fe) Bazı Biyokimyasal Parametrelere Etkileri. KSÜ Doğa Bilimleri Dergisi, 20(3), 175-184.
Kumar, A., Pandey, R., and Siddiqi, N., 2019. Oxidative stress biomarkers of cadmium toxicity in mammalian systems and their distinct ameliorative strategyi. Journal of Applied Biotechnology & Bioengineering, 6, 126-135.
Kumar, K.B. and Khan, P.A., 1982. Peroxidase and polyphenol oxidase in excised ragi (Eleusine coracana cv. PR 202) leaves during senescence. Indian Journal of Experimental Biology, 20, 412-416.
Lenártová, V., K. Holovská and P. Javorski.,1998. The influence of mercury on the antioxidant enzyme activity of rumen bacteria Streptococcus bovis and Selenomonas ruminantium. FEMS Microbiology Ecology, 27, 319-325.
Ligor, M., Ligor, T., Gadzała‐Kopciuch, R. and Buszewski, B., 2015. The chromatographic assay of 4‐hydroxynonenal as a biomarker of diseases by means of MEPS and HPLC technique. Biomedical Chromatography, 29, 584-589.
Lowry, O.H., Rosenbrough, N.J., Farr, A.L and Randall, R.J ., 1951. Protein measurement with the Folin-phenol reagent. The journal of Biochemistry, 193, 265- 277.
Manca, D., Ricard, A.C., Trottier, B. and Chevalier, G.,1991. Studies On Lipid Peroxidation in Rat Tissues Following Administration of Low and Moderate Doses of Cadmium Chloride. Toxicology, 67, 303–23.
Marklund, S. and Marklund, G., 1974. Involvement of the Superoxide Anion Radical in the Autoxidation of Pyrogallol and a Convenient Assay for Superoxide Dismutase. European Journal of Biochemistry, 47(3), 469–474.
O'Hara, C.M., Westbrook, G.L. and Miller, J.M., 1997. Evaluation of Vitek GNI+ and Becton Dickinson Microbiology Systems Crystal E/NF identification systems for identification of members of the family Enterobacteriaceae and other gram-negative, glucose-fermenting and non-glucose-fermenting bacilli. Journal of Clinical Microbiology, 35, 3269-3273.
Paglia, D.E. and Valetine, W.N., 1967. Studies on the quantitative and qualitative characterization of erytrocyte glutathione peroxidase. The Journal of Laboratory and Clinical Medicine, 70,158-169.
Pandey, S., Barai, P.K. and Maiti, T.K., 2013. Influence of heavy metals on the activity of antioxidant enzymes in the metal resistant strains of Ochrobactrum and Bacillus sp., Journal of environmental biology, 34, 1033-1037.
Paschal, D., Burt, V., Caudill, S., Gunter, E. W., Pirkle, J.L., Sampson, E. J., Miller, D.T. and Jackson, R.J., 2000. Exposure of the US population aged 6 years and older to cadmium, 1988–1994. Archives of environmental contamination and toxicology, 38, 377-383.
Puchenkova, S., 1996. Enterobacteria in areas of water along the Crimean Coast. Mikrobiolohichnyi Zhurnal (Kiev, Ukraine, 1993), 58, 3-7.
Sáez, G.T. and Están-Capell, N., 2017i Antioxidant Enzymes, in Encyclopedia of Cancer, M. Schwab, Editor Springer Berlin Heidelberg: Berlin, Heidelberg, 288-294.
Schaur, R.J., Siems, W., Bresgen, N. and Eckl, P.M., 2015. 4-Hydroxy-nonenal—a bioactive lipid peroxidation product. Biomolecules, 5, 2247-2337.
Shacter, E., 2000. Quantification and Significance of Protein Oxidation in Biological Samples. Drug Metabolism Reviews, 32(3&4), 307-326.
Smirnova, G. and Oktyabrsky, O., 2005. Glutathione in bacteria. Biochemistry (Moscow), 70, 1199-1211.
Taysi, S., 2005. Oxidant/Antioxidant Status In Liver Tissue Of Vitamin B6 Deficient Rats. Clinical Nutrition, 24, 385–9.
Vlasova, I.I. (2018). Peroxidase activity of human hemoproteins: keeping the fire under control. Molecules, 23, 2561.
Wang, J., Chang, S., Chen, Y. and Luh, K., 2000. Comparison of antimicrobial susceptibility of Citrobacter freundii isolates in two different time periods. Journal of Microbiology, Immunology and Infection, 33, 258-262.
Yerli, C., Çakmakci, T., Şahin, Ü. and Tüfenkçi, Ş., 2020. Ağır Metallerin Toprak, Bitki, Su ve İnsan Sağlığına Etkileri. Türk Doğa ve Fen Dergisi, 9, 103-114.
Zhang JF, Liub H, Sun YY, Wang XR, Wu JC, Xue, YQ 2005. Responses of the antioxidant defenses of the Goldfish Carassius auratus, exposed to 2,4- dichlorophenol. Environmental Toxicology and Pharmacology, 19, 185–190.