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Environmental Toxicology and Pharmacology 25 (2008) 144–147
Dechlorination of PCB in the presence of plant nitrate reductase Kristie D. Magee a , A. Michael b , H. Ullah a , S.K. Dutta a,∗ a
Molecular Genetics Laboratory, Department of Biology, Howard University, 415 College Street, NW, Washington, DC 20059, USA b GRL Laboratories, Washington, DC, USA Available online 13 October 2007
Abstract The dechlorination of PCB, specifically the noncoplanar congener PCB 153, has been observed in the presence of a crude nitrate reductase extract from Medicago sativa leaves. These observations were further confirmed using a commercially available and pure nitrate reductase from Zea mays. The presence of nitrate reductase increased PCB 153 dechlorination. Then, the addition of molybdenum, the enzyme’s cofactor, enhanced dechlorination of the environmental contaminant. The ability of plant nitrate reductase to dechlorinate PCB is a new observation. © 2007 Published by Elsevier B.V. Keywords: PCB; Dechlorination; Nitrate reductase; Medicago sativa
1. Introduction Neurotoxicity, endocrine disruption, reproductive abnormalities, and cancer are some of the adverse health effects that polychlorinated biphenyls (PCBs) can cause (Environmental Protection Agency, 2007; Safe, 1994; Carpenter, 1998). Degradation of PCBs is generally a two-step process: Chlorine atoms are reductively removed in anaerobic conditions, and the phenyl rings are oxidatively cleaved in aerobic conditions. The latter step is more familiarly known as the biphenyl degradation pathway, and many of the dioxygenases involved, as well as the genetic organization of the catabolic genes, have been elucidated (Iwasaki et al., 2006; Furukawa et al., 1989). However, the reductive enzymes and genes involved in the former dechlorination step are virtually unknown. We have previously observed the dechlorination of hexachlorobiphenyl by the white-rot fungus Phanerochaete chrysosporium. In this fungus, we identified a DNA sequence that is homologous to the nitrate reductase gene (De et al., 2006a). We are currently investigating whether or not a similar phenomenon is applicable to plants, for phytoremediation of halogenated compounds is well-documented by several sources (Mezzari et al., 2005; Van Aken et al., 2004; Yoon et al., 2006). Specifically, we are observing the relationship of a nitrate reductase gene, nia 1, in Medicago sativa (GenBank acces-
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1382-6689/$ – see front matter © 2007 Published by Elsevier B.V. doi:10.1016/j.etap.2007.10.009
sion number, AY584246) and its nitrate reductase product to the dechlorination of PCB 153 (2,2 ,4,4 ,5,5 -chlorobiphenyl). PCB 153 has been regarded as a model compound for the different structural classes of PCBs, which is why we decided to work with it (Lyche et al., 2004). While constitutive mRNAs of nia1 do exist, transcription of the gene primarily occurs upon exposing the plant to nitrate (its substrate) and light and results in the nitrate reductase enzyme (Beevers et al., 1965; Lewis, 1995; Sawhney and Naik, 1990). Nitrate reductase plays a role in nitrate assimilation of plants, some bacteria, and some fungi by reducing nitrate to nitrite. In plants, it is a cytoplasmic enzyme that employs several electron-transfer mediators (i.e. riboflavin, molybdenum ions, and NADH/NADPH). Another enzyme, nitrite reductase, completes nitrate assimilation by converting nitrite to ammonium that can be utilized for protein building (Ingle et al., 1966). In this study, we show the dechlorination of PCB 153 in the presence of a crude nitrate reductase extract from Medicago sativa leaves. We further confirm our observations using a pure, commercially available nitrate reductase from Zea mays (Sigma, St. Louis, MO). 2. Materials and methods 2.1. Plant material and in vitro reactions M. sativa seeds were germinated for 1 day before transferring to soil. Plants were grown for approximately 8 weeks in a controlled growth chamber (Conviron, Ltd., Pembina, ND) at a temperature of 25 ◦ C with a 14 h light/8 h dark photoperiod and watered regularly with tap water.
K.D. Magee et al. / Environmental Toxicology and Pharmacology 25 (2008) 144–147
Plants were subjected to 24 h of light in the presence or absence of 20 mM KNO3. Leaves were subsequently harvested and ground using a cold mortar and pestle in a grinding buffer comprised of 50 mM MOPS (pH 7.5), 10 mM MgCl2 , 1 mM EDTA, 0.5 mg/mL DTT, and 0.1% Triton X-100 as described by Rawls (www.uky.edu/∼jrawls/bio410/LAB%20MANUALnri.pdf, September 29, 2004). The homogenate was filtered through one layer of miracloth to obtain a crude enzyme extract, which was used for in vitro reactions comprised of 0, 15, or 50 L of extract, and 50 L NADH (in some cases, NADH omitted). In control reactions, grinding buffer and H2 O were used as substitutions for extract and NADH, respectively. Twenty-five microliters of PCB 153 and standard assay solution (25 mM phosphate buffer at pH 7.5 and 10 mM KNO3 ) were added to all reactions. There were duplicates for each reaction, incubation occurred for 10 min at 30 ◦ C, and Zn acetate was used to stop each reaction. In vitro 1 mL reactions using a nitrate reductase from Z. mays (Sigma) consisted of 25 mM phosphate buffer (with 10 mM KNO3 and 0.05 mM EDTA), 2 mM NADH, 50 ppm of PCB 153, and for enhancement conditions, 3 mM Na2 MoO4 . Enzyme activity was 0.02 units/mL; pH was 7.3. After 10 min, reactions were stopped by placing samples in a boiling hot water bath. Experiments were repeated at least three times.
2.2. Nitrate reductase activity A colorimetric nitrite release assay was used to determine nitrate reductase activity as described by Bergmann and Sanik (1957) using sulfanilamide in HCl and N-(1-naphthyl)-ethylenediamine hydrochloride at a wavelength of 540 nm. Analysis of samples was done in duplicates.
2.3. Gas chromatography–mass Spectrometry (GC–MS) PCB 153 was first extracted from each in vitro sample by addition of an equal volume of a hexane:acetone (1:1) mixture and vigorous shaking. The upper layer was removed and transferred into a clean vial. GC–MS was done using Agilent 6890N interfaced with an Agilent 5973 inert Mass Spectra following methods of De et al. (2006b). Biphenyl (BPH) was used as an internal standard in the extraction of PCB. The PCB/BPH ratios were obtained according to the area percentage of the peaks and were used to assess the degradation of PCB. BPH retention time was 6.84 and 10.13, respectively.
2.4. Fourier-transformed infrared spectroscopy (FTIR) Liquid samples from in vitro reactions were directly analyzed by the FTIR apparatus (GRL Laboratories, Washington, DC). Twenty-five scans were taken and averaged to obtain spectra between 1500 and 650 cm−1 . For qualitative FTIR analysis, the existence of the carbon–chlorine (C–Cl) functional group by the presence of its specific functional group peak was determined. A spectrum of PCB 153 dissolved in methanol at 1000 ppm was used to identify the C–Cl region and became a standard for all other spectra (Fig. 1b). For quantitative FTIR analysis, the strength (absorbance) of the absorption band was measured. Absorbance is proportional to the amount of the functional group (JASCO, Inc., Easton, MD).
Fig. 1. (a) PCB/BPH ratios after incubation in the presence or absence of varying amounts of M. sativa crude nitrate reductase extracts. Data shown represent one experiment. (b) Standard FTIR spectrum of PCB 153 showing C–Cl region at 820.31 cm−1 . Absorbance values obtained from the y-axis of respective FTIR spectra. (c) Dechlorination of PCB 153 after incubation in varying treatments of Z. mays nitrate reductase. While several FTIR analyses were done, the data shown represent one typical experiment.
Table 1 The correlation of NO2 − production to M. sativa crude nitrate reductase extract amounts from KNO3 -treated plants and plants not treated with KNO3
3.1. Nitrate reductase activity
Plant extracta (L)
The production of NO2 − was initially observed to assess activity of M. sativa crude nitrate reductase extract from plants that were treated with KNO3 as well as from those not treated with KNO3 (Table 1). There generally was a decrease in NO2 − with a decrease in crude extract amount in both sets of plants. The values were 63.9 ± 0, 64.1 ± 0.1, and 62.8 ± 0 for 50, 50 (no NADH), and 15 L of extract from KNO3 -treated plants, respectively. Control values (0 L of extract and 0 L of extract
Release of nitrite (NO2 − ) in treatment groups (nmol/mL) Nitrate (NO3 − )
0 0 (no NADHb ) 15 50 50 (no NADHb ) a b
62.0 60.9 62.8 63.9 64.1
± ± ± ± ±
0.2 0.1 0 0 0.1
Crude extracts containing nitrate reductase. 50 L/ mL of NADH.
No Nitrate (NO3 − ) 61.1 61.0 62.0 62.8 62.0
± ± ± ± ±
0.1 0 0 0.45 0
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minus NADH) were 62.0 ± 0.2 and 60.9 ± 0.1 nmol NO2 − , respectively. For plants that were not treated with KNO3 , the values were 62.8 ± 0.45 (50 L extract), 62.0 ± 0 (50 L extract minus NADH), and 62.0 ± 0 (15 L extract). Values for controls were 61.1 ± 0.1 (0 L extract) and 61.0 ± 0 (0 L extract minus NADH) nmol NO2 − . The production of NO2 − from plants that were not treated with KNO3 was lower in every case (with the exception of 0, no NADH) in comparison to plants that were treated with KNO3 (Table 1). 3.2. GC–MS analysis The effects of incubation on PCB 153 amounts after incubation in the presence or absence of M. sativa crude nitrate reductase extracts from KNO3 -treated plants and plants not treated with KNO3 were analyzed by GC–MS (Fig. 1a). PCB/BPH ratios decreased with an increase in crude extract amounts from KNO3 -treated plants; ratios were 1.921, 1.271, and 0.341 for 0, 15, and 50 (no NADH) L, respectively. For plants not treated with KNO3 , no consistent relationship between the decrease of PCB 153 and nitrate reductase extract amounts was observed. PCB/BPH ratios were 0.872 (0 L), 0.623 (15 L), and 0.798 (50 L, no NADH). 3.3. FTIR analysis To further confirm the GC–MS results, the effects of incubation on PCB 153 dechlorination in the presence or absence of Z. mays nitrate reductase was observed and analyzed via FTIR (Fig. 1c). In control samples devoid of nitrate reductase, the C–Cl peak was evident at 830.48 cm−1 (Fig. 1b used as a reference) and had an absorbance of 0.68. In samples containing nitrate reductase, the absorbance of the C–Cl region decreased from 0.68 to 0.56. The absorbance of the C–Cl region decreased even further upon the addition of molybdenum, a cofactor of nitrate reductase, to 0.15. 4. Discussion In M. sativa, the amount of crude enzyme extract generally correlated to the release of NO2 − : The lesser the extract amount, the lesser NO2 − produced. While KNO3 -treated plants yielded greater NO2 − amounts than plants not treated with KNO3 , the disparities were negligent. The GC–MS and FTIR data indicated that plant nitrate reductase was capable of dechlorinating PCB 153. Crude M. sativa nitrate reductase extract amounts and PCB/BPH ratios were, for the most part, inversely proportional. Similarly, Z. mays nitrate reductase activity was inversely proportional to the absorbance of PCB 153’s C–Cl region. The presence and absence of nitrate reductase did affect dechlorination, with its presence increasing dechlorination of PCB 153. Then, the addition of molybdenum, the cofactor of nitrate reductase, enhanced PCB 153 dechlorination. Of course, the dechlorination of PCB 153 by nitrate reductase carries with it the notion that the enzyme is capable of binding to
the aromatic compound. While the primary substrate of nitrate reductase is NO3 − , this notion does not seem to be unreasonable, for the enzyme has demonstrated flexibility in terms of its specificity. The capacity of membrane-bound nitrate reductases to reduce various substrates, such as bromate, chlorate, selenate, and tellurite, is a well-known phenomenon (Avazeri et al., 1997; Sabaty et al., 2001). Also, the reduction of lindane being dependent on the function of the nir operon, which codes for nitrate reductase in Anabaena sp., is further exemplary of the enzyme’s widespread specificity (Kuritz et al., 1997). In conclusion, we have shown the dechlorination of PCB 153 in the presence of a crude nitrate reductase extract from Medicago sativa leaves. We further confirmed our observations using a commercially available and pure nitrate reductase from Zea mays. This study is parallel to our previous findings with P. chrysosporium and PCB 153. The dechlorination of the hazardous environmental contaminant by plant nitrate reductase is a new observation. Future studies are directed toward comparing the expression of nia1 by real-time RT-PCR to the dechlorination of PCB 153 in various conditions. Acknowledgements The authors thank Dr. Somiranjan Ghosh for technical support as well as the HU Graduate School and NIH/SCORE projects to SKD for financial assistance. References Avazeri, C., Turner, R.J., Pommier, J., Weiner, J.H., Giordano, G., Vermeglio, A., 1997. Tellurite reductase activity of nitrate reductase is responsible for the basal resistance of Escherichia coli to tellurite. Microbiology 43, 1181–1189. Beevers, L., Schrader, L., Flesher, D., Hageman, R., 1965. The role of light and nitrate in the induction of nitrate reductase in radish cotyledons and maize seedlings. Plant Physiol. 40, 691–698. Bergmann, J.G., Sanik, J., 1957. Determination of trace amounts of chlorine in naphta. Anal. Chem. 29, 241–243. Carpenter, D.O., 1998. Polychlorinated biphenyls and human health. Int. J. Occup. Med. Environ. Health 11, 291–303. De, S., Ghosh, S., Dutta, S.K., 2006b. Congener specific polychlorinatedbiphenyl metabolism by human intestinal microbe Clostridium species: comparison with human liver cell line-HepG2. Ind. J. Microbiol. 46, 191–199. De, S., Perkins, M., Dutta, S.K., 2006a. Nitrate reductase gene involvement in hexachlorobiphenyl by Phanerochaete chrysosporium. J. Hazard. Mater. B 135, 350–354. Environmental Protection Agency, 2007. Health Effects of PCBs. Available www.epa.gov/pcb/pubs/effects.htm, October 10, 2007. Furukawa, K., Hayase, N., Taira, K., Tomizuka, N., 1989. Molecular relationship of chromosomal genes encoding biphenyl/polychlorinated biphenyl catabolism: some soil bacteria possess a highly conserved bph operon. J. Bacteriol. 171, 5467–5472. Ingle, J., Joy, K.W., Hageman, R.H., 1966. The regulation of activity of the enzymes involved in the assimilation of nitrate by higher plants. Biochem J. 100, 577–588. Iwasaki, T., Miyauchi, K., Masai, E., Fukuda, M., 2006. Multiple-subunit genes of the aromatic-ring-hydroxylating dioxygenase play an active role in biphenyl and polychlorinated biphenyl degradation in Rhodococcus sp. strain RHA1. Appl. Environ. Microbiol. 72 (8), 5396–5402. Kuritz, T., Bocanera, L., Rivera, N., 1997. Dechlorination of lindane by the cyanobacterium Anabaena sp. strain PCC7120 depends on the function of the nir operon. J. Bacteriol. 179, 3368–3370.
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