Effects of Polychlorinated Biphenyls (PCBs) on Root Meristem Cells of Common Onion (Allium cepa L.) and on Early Life Stages of Zebrafish (Danio rerio)
Jonas P. Quilang1*, Margaret C. de Guzman1, Maria Helen de Hitta-Catalan1,2,
Raquel O. Rubio3, Sonia D. Jacinto1, Evangeline C. Santiago3, and Ernelea P. Cao1,3
1Institute of Biology, College of Science, University of the Philippines,
Diliman, Quezon City, Philippines
2National Institute for Science and Mathematics Education Development
University of the Philippines, Diliman, Quezon City, Philippines
3Natural Sciences Research Institute, University of the Philippines
Diliman, Quezon City, Philippines
Polychlorinated biphenyls (PCBs) are chemical mixtures that are common environmental pollutants and considered potential carcinogens. In this study, the common onion, Allium cepa, was used to determine the cytotoxicity and genotoxicity of a mixture of PCBs at three concentrations, namely, 10 nM, 100 nM, and 1000 nM. Distilled deionized water and 0.2% dimethylsulfoxide (DMSO) were used as negative controls. Cytotoxicity was observed at the three concentrations tested as shown by a significant reduction in average mitotic indices. Chromosomal aberrations were also observed, which is suggestive of the genotoxicity of these pollutants. The effects of PCBs on the early life stages of zebrafish, Danio rerio, were also investigated. The three levels of PCBs were tested including the embryo medium, 0.2% DMSO, and 2% ethanol that served as controls. Embryos at 6 – 8 hours postfertilization were exposed to the different treatments and were observed everyday for 5 days. Results showed a significant decrease in the mean number of melanocytes and average length at 5 days postfertilization, a delay in hatching of embryos, and a significant increase in average percent of fish with deformities for the 1000 nM PCB concentration. This study demonstrates that the Allium test is a simple and reliable method to assess cytotoxicity and genotoxicity of PCBs. Exposure of zebrafish embryos to high concentrations of PCBs also delays growth and hatching rates and causes teratogenic effects. These results may have implications on the health of humans and other organisms at risk to PCB exposures as these chemicals are known to bioaccumulate.
Polychlorinated biphenyls (PCBs) are a group of synthetic organic chemicals that are formed by adding chlorines to biphenyl rings. Since a biphenyl ring has 10 possible positions for chlorine substitution, theoretically, there are 209 possible arrangements or congeners, but only about 60 to 90 congeners are present in commercial mixtures (Kimbrough & Krouskas 2003). PCBs were commercially produced in the US from 1929 to 1977 with the trade names Aroclor 1016, 1242, 1254, 1260, and 1268 (Kimbrough & Krouskas 2003). In the past, commercial PCB mixtures were used in capacitors and transformers, flame retardants, inks, adhesives, microencapsulation of dyes for carbonless duplicating paper, paints, pesticide extenders, plasticizers, polyolefin catalyst carriers, surface coatings, wire insulators, and metal coatings (Kimbrough & Krouskas 2003). Owing to accumulating evidence that they build up in the environment and have harmful effects, production of PCB mixtures has been stopped in most developed countries. But it is unknown if these are still being produced in other countries.
APOSTOLI P, MAGONI M, BERGONZI R, CARASI S, INDELICATO A, SCARCELLA C, DONATO F. 2005. Assessment of reference values for polychlorinated biphenyl concentration in human blood. Chemosphere 61: 413-421.
[ATSDR] Agency for Toxic Substances and Disease Registry. 2000. Toxicological profile for polychlorinated biphenyls (PCBs). Atlanta, Georgia, USA: U.S. Department of Health and Human Services, Agency for Toxic Substances and Disease Registry. 948p.
ATEEQ B, ABUL FARAH M, NIAMAT ALI M, AHMAD W. 2002. Clastogenicity of pentachlorophenol, 2,4- D and butachlor evaluated by Allium root tip test. Mutation Res 514: 105-113.
BILLSSON K, WESTERLUND L, TYSKLIND M, OLSSON P-E. 1998. Developmental disturbances caused by polychlorinated biphenyls in zebrafish (Brachydanio rerio). Marine Environ Res 46: 461-464.
BLACK DE, PHELPS DK, LAPAN RL. 1988. The effect of inherited contamination on egg and larval winter flounder, Pseudopleuronectes americanus. Marine Environ Res 25: 45-62.
BUTTERWORTH FM, PANDEY P, MCGOWEN RM, ALI-SADAT S, WALIA S. 1995. Genotoxicity of polychlorinated biphenyls (PCBs): recombinogenesis by biotransformation products. Mutation Res 342: 61-69.
CHANDRA S, CHAUHAN LKS, MURTHY RC, SAXENA PN, PANDE PN, GUPTA SK. 2005. Comparative biomonitoring of leachates from hazardous solid waste of two industries using Allium test. Sci Total Environ 347: 46-52.
CHENG SH, WAI AWK, SO CH, WU RSS. 2000. Cellular and molecular basis of cadmium-induced deformities in zebrafish embryos. Environ Toxicol Chem 19: 3024-31.
CONCEPCION MTA. 2006. Monitoring exposure to PCBs located at the Meralco Pasig central service station. International POPs Elimination Project Report (http://www.ipen.org). Accessed 06 November 2007.
CREBELLI R, CONTI L, MONARCA S, FERETTI D, ZERBINI I, ZANI C, VESCHETTI E, CUTILLI D, OTTAVIANI M. 2005. Genotoxicity of the disinfection by-products resulting from peracetic acid- or hypochlorite-disinfected sewage wastewater. Water Res 39: 1105-13.
DELTOUR L, ANG HL, DUESTER G. 1996. Ethanol inhibition of retinoic acid synthesis as a potential mechanism for fetal alcohol syndrome. FASEB J 10: 1050-57.
ELONEN GE, SPEHAR RL, HOLCOMBE GW, JOHNSON RD, FERNANDEZ JD, ERICKSON RJ, TIETGE JE, COOK PM. 1998. Comparative toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin to seven freshwater fish species during early life-stage development. Environ Toxicol Chem 17: 472-483.
EL-SHAHABY OA, ABDEL MIGID HM, SOLIMAN MI, MASHALY IA. 2003. Genotoxicity screening of industrial wastewater using the Allium cepa chromosome aberration assay. Pakistan J Biol Sci 6: 23-28.
EVANDRI MG, BOLLE P. 2001. Pharmaco-toxicological screening of commercially available Italian natural mineral waters. Il Farmaco 56: 475-482.
FISKESJO G. 1985. The Allium test as a standard in environmental monitoring. Hereditas 102: 99-112.
FONT G, MANES J, MOLTO JC, PICO Y. 1996. Current developments in the analysis of water pollution by polychlorinated biphenyls. J Chromatography A 733: 449-471.
GRANT WF. 1982. Chromosome aberration assays in Allium: A report of the U.S. Environmental Protection Agency Gene-Tox Program. Mutation Res 99: 273-291.
GROVER IS, KAUR S. 1999. Genotoxicity of wastewater samples from sewage and industrial effluent detected by the Allium root anaphase aberration and micronucleus assays. Mutation Res 426: 183-188.
HALLARE AV, KOHLER H-R, TRIEBSKORN R. 2004. Developmental toxicity and stress protein responses in zebrafish embryos after exposure to diclofenac and its solvent, DMSO. Chemosphere 56: 659-666.
HAHN ME. 2001. Dioxin toxicology and the aryl hydrocarbon receptor: insights from fish and other nontraditional models. Marine Biotechnol 3: S224-S238.
HERRMANN K. 1993. Effects of the anticonvulsant drug valproic acid and related substances on the early development of the zebrafish (Brachydanio rerio). Toxicol in vitro 7: 41-54.
JÖNSSON ME, JENNY MJ, WOODIN BR, HAHN ME, STEGEMAN J. 2007. Role of AHR2 in the expression of novel cytochrome P450 1 family genes, cell cycle genes, and morphological defects in developing zebra fish exposed to 3,3’,4,4’,5-pentachlorobiphenyl or 2,3,7,8-tetrachlorodibenzo-p-dioxin. Toxicol Sci 100(1): 180-193.
KIMBROUGH RD, KROUSKAS CA. 2003. Human exposure to polychlorinated biphenyls and health effects: a critical synopsis. Toxicol Rev 22: 217-233.
LOCKWOOD B, BJERKE S, KOBAYASHI K, GUO S. 2004. Acute effects of alcohol on larval zebrafish: a genetic system for large-scale screening. Pharmacol Biochem Behav 77: 647-654.
MATSUI JI, EGANA AL, SPONHOLTZ T, ADOLPH AR, DOWLING JE. 2006. Effects of ethanol on photoreceptors and visual function in developing zebrafish. Invest Ophthalmol Vis Sci 47(10): 4589-97.
MATTA MB, CAIRNCROSS C, KOCAN RM. 1997. Effect of a polychlorinated biphenyl metabolite on early life stage survival of two species of trout. Bull Environ Contam Toxicol 59: 146-151.
OLSSON P-E, WESTERLUND L, TEH SJ, BILLSSON K, BERG AH, TYSKLIND M, NILSSON J, ERIKSSON L-O, HINTON DE. 1999. Effects of maternal exposure to estrogen and PCB on different life stages of zebrafish (Danio rerio). Ambio 28: 100-106.
RANK J, NIELSEN MH. 1997. Allium cepa anaphasetelophase root tip chromosome aberration assay on N-methyl-N-nitrosourea, maleic hydrazide, sodium azide, and ethyl methanesulfonate. Mutation Res 390: 121-127.
SAMSON JC, SHENKER J. 2000. The teratogenic effects of methylmercury on early development of the zebrafish, Danio rerio. Aquatic Toxicol 48: 343-354.
SINHA P, KANAMADI R. 2000. Effect of mercurial fungicide Emisan®-6 on the embryonic, developmental stages of zebrafish, Brachydanio (Danio) rerio. J Adv Zool 21: 12-18.
SMAKA-KINCL V, STEGNAR P, LOVKA M, TOMAN MJ. 1996. The evaluation of waste, surface and ground water quality using the Allium procedure. Mutation Res 368: 171-179.
TANABE S, TATSUKAWA R. 1987. Mussels as bioindicators of PCB pollution: A case study on uptake and release of PCB isomers and congeners in greenlipped mussels (Perna viridis) in Hong Kong waters. Environ Pollut 47: 41-62.
TERAOKA H, DONG W, OGAWA S, TSUKIYAMA S, OKUHARA Y, NIIYAMA M, UENO N, PETERSON RE, HIRAGA T. 2002. 2,3,7,8-tetrachlorodibenzo-pdioxin toxicity in the zebrafish embryo: altered regional blood flow and impaired lower jaw development. Toxicol Sci 65: 192-199.
TIEDEKEN JA, RAMSDELL JS, RAMSDELL AF. 2005. Developmental toxicity of domoic acid in zebrafish (Danio rerio). Neurotoxicol Teratol 27: 711-717.
TODD NE, VAN LEEUWEN M. 2002. Effects of Sevin (carbaryl insecticide) on early life stages of zebrafish (Danio rerio). Ecotoxicol Environ Safety 53: 267-272.
UENO D, INOUE S, IKEDA K, TANAKA H, YAMADA H, TANABE S. 2003a. Specific accumulation of polychlorinated biphenyls and organochlorine pesticides in Japanese common squid as a bioindicator. Environ Pollut 125: 227-235.
UENO D, TAKAHASHI S, TANAKA H, SUBRAMANIAN AN, FILLMAN G, NAKATA H, LAM PKS, ZHENG J, MUCHTAR M, PRUDENTE M, CHUNG KH, TANABE S. 2003b. Global pollution monitoring of PCBs and organochlorine pesticides using skipjack tuna as a bioindicator. Arch Environ Contam Toxicol 45: 378-389.
WESTERLUND L, BILLSSON K, ANDERSSON PL, TYSKLIND M, OLSSON P-E. 2000. Early life-stage mortality in zebrafish (Danio rerio) following maternal exposure to polychlorinated biphenyls and estrogen. Environ Toxicol Chem 19: 1582-88.
WHYSNER J, WANG C-X. 2001. Hepatocellular iron accumulation and increased cell proliferation in polychlorinated biphenyl-exposed Sprague-Dawley rats and the development of hepatocarcinogenesis. Toxicol Sci 62: 36-45.
WONG A, BASRUR P, SAFE S. 1979. The metabolically mediated DNA damage and subsequent DNA repair by 4-chlorobiphenyl in Chinese hamster ovary cells. Res Comm Chem Pathol Pharmacol 24: 543-550.
WYNDHAM C, DEVENISH J, SAFE S. 1976. The in vitro metabolism, macromolecular binding and bacterial mutagenicity of 4-chlorobiphenyl, a model PCB substrate. Res Comm Chem Pathol Pharmacol 15: 563-570.
WYNDHAM C, SAFE S. 1978. In vitro metabolism of 4-chlorobiphenyl by control and induced rat liver microsomes. Biochem 17: 208-215.