Environ. Sci. Technol. 2010, 44, 8891–8896
Inhibition of Free DNA Degradation by the Deformation of DNA Exposed to Trace Polycyclic Aromatic Hydrocarbon Contaminants F U X I N G K A N G , † Y A N Z H E N G G A O , * ,† A N D QIAN WANG‡ Institute of Organic Contaminant Control and Soil Remediation, College of Resource and Environmental Science, Nanjing Agricultural University, Nanjing 210095, P.R. China, and Nanjing Institute of Geography and Limnology, Chinese Academy of Sciences, Nanjing 210008, P.R. China
Received February 24, 2010. Accepted October 22, 2010.
A rapid inhibitory effect of polycyclic aromatic hydrocarbons (PAHs) on DNA degradation was examined by conventional spectral analysis and microtitration. The purpose was to determine whether PAHs inhibited free DNA degradation by the enzyme DNase I. The results showed that model PAHs phenanthrene and pyrene combined with free DNA to decelerate DNA degradation by DNase I. Phenanthrene-induced inhibition was stronger than that of pyrene. Trace level of PAHs did not induce DNase I deactivation. The DNase I enzyme exhibited only slight shifts in IR absorption bands related to amide II and III upon PAH exposure, and no change was observed with other bands. The decelerating degradation of DNA is attributed to the changes in structure, backbone composition, and guanine constituents of DNA induced by PAHs inserted into double strands, and to the imidazole-like derivates from the combination of imidazole rings with pyrene.
Introduction Free deoxyribonucleic acid (DNA) from biological cell disruption or extracellular secretion in soil and water columns plays an important role in biological heredity and variation, ecologic and genetic diversity, and biological evolution (1, 2). Generally, the ubiquitous bivalent ions in soil and/or water can assist the entry of these DNA segments to the interior of cells by changing the permeability of the cellular membrane (3-5). The resulting syncretic DNA, which may act as a new genetic character, can be imbibed into a receptor by horizontal gene transfer (HGT) thereby affecting the receptor’s biological character (1, 6, 7, 30). Recent reports have shown that up to 10-16% of E. coli DNA is from HGT (1, 8), which demonstrates that gene transfer between organisms, and especially between microorganisms, is ubiquitous. Generally, deoxyribonuclease I (DNase I) is released along with DNA into the environment. The widespread presence of a large number of bivalent cations, like Ca2+, Mg2+, and Mn2+, allow the DNA double strand to be randomly sheared by this enzyme. Thus, the released DNA is gradually degraded or immediately metabolized and is difficult to preserve in the environment. However, natural environmental factors * Corresponding author phone: +86-25-84395238; fax: +86-2584395210; e-mail:
[email protected]. † Nanjing Agricultural University. ‡ Chinese Academy of Sciences. 10.1021/es103215b
2010 American Chemical Society
Published on Web 11/05/2010
such as soil granules, temperature, and organic matter can affect this enzymatic degradation (2, 9). Earlier reports have also shown that clay minerals can adsorb the DNA segments and retard enzymatic degradation (10). This would allow the DNA segments in soil and/or water to remain for a relatively long time, thereby increasing the possibility of making contact with an acceptor. Accelerated anthropogenic activities including agriculture and industry have dramatically affected these natural processes. The exogenous products of these activities have been increasingly released into the atmosphere, soil, and water through the emission of exhaust gases, wastewater, and waste residues. It has become increasingly important to consider the full impact of these products, especially hazards like organic contaminants. Polycyclic aromatic hydrocarbons (PAHs) are a group of persistent organic contaminants that are highly toxic, carcinogenic, accumulate biologically, and are ubiquitous in the environment (11-13). Sources include the incomplete decomposition of organic residues, petrochemicals production, volcanic eruptions, and exudates from plants (14). Concentrations of PAHs in surface soils from certain regions of China range from 203.8 to 6753 µg · kg-1 (15). A study on the distribution of PAHs in some rivers of China indicated that the total PAH concentrations in the water ranged from 21.7 to 138 µg · L-1, and that concentrations in suspended particulates were from 40.9 to 238 µg · kg-1 (16). Although PAHs are inherently hydrophobic and exhibit low water solubility, the abundance of suspended mineral and organic materials allows more these contaminants to remain in the water column. Previous reports have shown that PAHs in the cell interior can induce changes in DNA sequence, three-dimensional structure, and the expression of the germplasm, due to their biological affinity (17-19). Few reports have examined the influence of PAHs, especially trace PAH contaminants, on the degradation of extracellular free DNA segments with respect to the background of accelerating anthropogenic activities. In the present study, both free DNA damage induced by trace amounts of PAHs and the influence of such deformations on DNA degradation were explored using a series of spectroscopic tools and microtitration methods. The results obtained from this study will provide insight into the structural deformation and degradation of extracellular DNA exposed to PAH contaminants and strengthen our understanding of how anthropogenic trace PAHs affect and/ or threaten biological heredity and variation, ecologic and genetic diversity, and biological evolution.
Experimental Section Reagents. Phenanthrene and pyrene were purchased from Aldrich Chemical Co. Ltd.; their physicochemical properties are given in Supporting Information (SI) Table S1. DNA from salmon sperm and DNase I enzyme were purchased from the Shanghai Ruicong Scientific and Technological Co. Ltd. DNA purity was assessed by measuring the OD260/OD280 ratio (1.908). During DNA degradation experiments, a DNA reactive solution composed of bivalent calcium ion (CaCl2) at a concentration of 10 mmol · L-1 was prepared to activate the DNase I enzyme and to preserve the configuration of DNA in aqueous solution. Interactions of PAHs with DNA. The influences of PAHs on DNA bases and structure were determined by microtitration. A stock solution of 100 mg · L-1 DNA was prepared. Aqueous phenanthrene and pyrene solutions (100 µg · L-1) were prepared by diluting methanolic stock solutions of 2000 mg · L-1 phenanthrene and pyrene, respectively. The diluted VOL. 44, NO. 23, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 1. The temporal remnant concentration of DNA degraded by DNase at different PAH concentration gradient at 25 (Left: phenanthrene; Right: pyrene). The concentrations of DNA and DNase I were 600 and 170 µg · L-1, respectively. The fitting equations of temporal remnant DNA were listed as follows. Without any PAHs: y ) 383 + 216 × 10(-t/2656), R2 ) 0.845; 0.05 µg · L-1 phenanthrene: y ) 552 + 48 × 10(-t/182), R2 ) 0.583; 0.10 µg · L-1 phenanthrene: y ) 592 + 8 × 10(-t/191), R2 ) 0.415; 0.1 µg · L-1 pyrene: y ) 572 + 34 × 10(-t/256), R2 ) 0.606; 0.3 µg · L-1 pyrene: y ) 592 + 6 × 10(-t/226), R2 ) 0.456. DNA solution was gradually titrated into the aqueous phenanthrene/pyrene solutions using a chromatographic injector with a scale of 10 µL. After each addition of DNA, the fluorescent intensity was recorded and a spectrum of phenanthrene/pyrene was immediately collected at a fixed excitation wavelength using an enzyme-labeled meter with a fluorescence resolution of
