Antioxidant Enzyme Activity in Bacterial Resistance to Nicotine Toxicity by Reactive Oxygen Species
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- Shao, T., Yuan, H., Yan, B. et al. Arch Environ Contam Toxicol (2009) 57: 456. doi:10.1007/s00244-009-9305-z
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We analyzed superoxide dismutase (SOD), catalase (CAT), and ATPase activities in the highly nicotine-degrading strain Pseudomonas sp. HF-1 and two standard strains Escherichia coli and Bacillussubtilis in an attempt to understand antioxidant enzymes in bacteria are produced in response to nicotine, which increases the virulence of the bacteria. Nicotine had different effects on different antioxidant enzymes of different bacteria. SOD plays a more important role in resistance to nicotine stress in E. coli than it does in CAT. Multiple antioxidant enzymes are involved in combating oxidative stress caused by nicotine in Pseudomonas sp. HF-1. The contribution of a particular antioxidant enzyme for protection from nicotine stress varies with the growth phase involved. The inhibition of ATPase in Pseudomonas sp. HF-1 at the stationary phase was enhanced with increasing nicotine concentration, showing a striking dose–response relationship. Nicotine probably affected the metabolism of ATP to some extent. Furthermore, different bacteria possessed distinct SOD isoforms to cope with oxidative stress caused by nicotine.
Nicotine ([1-methyl-2-(3-pyridyl)] pyrrolidine), a major alkaloid synthesized as an L-isomer, is a significant toxic waste product in tobacco production. The tobacco-manufacturing process and all activities that use tobacco produce solid and liquid waste with high concentrations of nicotine (Novotny and Zhao 1999). Meanwhile, nicotine is a developmental neurotoxin that is thought to generate oxidative radicals (Qiao et al. 2005) and affect a variety of cellular processes ranging from induction of gene expression to secretion of hormones and regulation of enzymatic activity (Yildiz et al. 1998). The effect of nicotine on organisms has not been completely elucidated to date. It was illustrated that iminium metabolites, via electron transfer (ET) with redox cycling, might be involved in the production of radical entities. Free radicals are widely involved in many intracellular processes. Low levels of radicals appear to benefit the metabolic processes of living organisms, whereas high concentrations are associated with toxicity (Kovacic and Cooksy 2005).
Previous studies have shown that many pollutants are redox active and that these redox-cycling chemicals are able to enter microorganisms and influence aerobic metabolism, which causes a univalent reduction of molecular oxygen, leading to the superoxide radical H2O2 and the hydroxyl radical (Kang et al. 2007; Kreiner et al. 2002) . On the other hand, pollutant-degrading bacteria might experience oxidative stress, both as a direct effect of the pollutants themselves and from intermediates generated during the biodegradation processes (Park et al. 2004). The reactive oxygen species (ROS) can be damaging for bacterial cells, whereas the toxic effects of ROS can be minimized by antioxidant enzymes such as superoxide dismutases (SODs), catalase (CAT), and glutathione peroxidase (GSH-Px). SOD can dismutate the superoxide anion (O2–) to H2O2 and O2 (Gerlach et al. 1998) and then H2O2 is eliminated by the H2O2-scavenging enzyme CAT (Hidalgo et al. 2004; Jung 2003; Loprasert et al. 1996). Meanwhile, ATPase plays an important role in many intracellular physiological functions and can be considered a sensitive indicator of toxicity (Yadwad et al. 1990).
Pseudomonas sp. HF-1, isolated from nicotine-contaminated soil, can utilize nicotine as its sole carbon and energy source. Our results showed that nicotine had different effects on different antioxidant enzymes of different bacteria. So far, the oxidative stress responses to several pollutants have been extensively examined in bacteria (Hassett et al. 2000; Geckil et al. 2003; Frederick et al. 2001). The impacts of nicotine on oxidative stress also have been largely investigated in mammiferous cells (Elkins et al. 1999; Kane et al. 2004; Lü et al. 2004; Diaz et al. 2000). However, no previous studies have investigated the oxidative stress response of common microorganisms to nicotine. This study aimed to provide such information and determine the role of SOD, CAT, and ATPase in the protection of bacteria against oxidative stress.
Materials and Methods
Strains and Growth Conditions
Nicotine-degrading strain HF-1 was isolated from the soil of the Hefei Cigarette Factory (China) and identified as Pseudomonas sp. The strain can grow with nicotine as the sole source of carbon and energy (Ruan et al. 2005). Gram-negative representative strain Escherichiacoli K12 and Gram-positive representative strain Bacillussubtilis B19 were used as comparative strains in the study.
To elucidate the toxicity and influence of nicotine to strain HF-1, the bacterial viability of strain HF-1 in the presence and absence of 1 g/l nicotine in Lurie–Bertani (LB) media were investigated by reading the optical density at a wavelength of 600 nm (OD600 nm) at certain intervals. For other enzyme activity experiments, the strains were inoculated into nicotine-containing LB media and incubated at 30°C with shaking at 150 rpm. LB media were prepared by phosphate buffer (pH 7.0) for excluding the pH shift caused by the addition of nicotine.
Exposition of Cells to Different Concentrations of Nicotine
In order to ascertain the maximal response reactive concentration, nicotine was added into growing Pseudomonas sp. HF-1 cells (nicotine added at the same time of inoculation) and grown Pseudomonas sp. HF-1 cells (nicotine added when the cells have been incubated for 24 h at 30°C) with final concentrations of 0, 0.5, 1.0, 1.3, and 2.0 g/l, respectively. All treated cultures were incubated at 30°C for 24 h before being harvested. Spectrophotometry readings of OD600 nm were taken at a certain interval.
Different Cells Exposed to Nicotine
Pseudomonas sp. HF-1, E. coli K12, and B. subtilis B19 were incubated in 150 mL LB medium containing a suitable concentration of nicotine according to the experimental results mentioned earlier. Nicotine was added at the beginning and at the early stationary phase of strain HF-1 growth. The intracellular enzyme activities were assayed at designed intervals.
Preparation of Crude Extracts
Fresh bacterial cultures were harvested by centrifugation at 6000 rpm for 15 min. The harvested cells were washed twice using ice-cold 0.9% sodium chloride solution and resuspended in 3 mL of the same solution. Suspensions were sonicated on ice for 99 cycling (intervals of 3 s working with 3 s of cooling between bursts) and then centrifuged at 10,000g for 10 min at 4°C for removal of unbroken cells and cell debris. The supernatant was stored at –20°C for further assays.
Enzyme Activity Assays
Total protein content in cell lysates was determined by a modified Lowry procedure (Kit A045) using bovine serum albumin as the reference. The catalase, ATPase, and SOD specific activities of cell lysates were detected using the spectrophotometric protocols with Kit A007, A016, and A001, respectively. All kits were purchased from Nanjing Jiancheng Bioengineering Institute (Jiangsu Province, China). One unit of CAT was defined as the amount of lysate that catalyzes the decomposition of 1 μmol of H2O2 per minute at 25°C. One unit of SOD activity was defined as the amount of lysate that inhibits the rate of xanthine/xanthine oxidase-dependent cytochrome-c reduction at 25°C by 50%. The specific activity was expressed as units per milligram of cellular proteins. One unit of ATPase activity was defined as the amount of inorganic phosphorus produced by ATP decomposition per hour per milligram of protein.
All data were expressed as mean ± S.E. of three replicates. The Student’s t-test was used to determine the significance level of differences between mean values. The indexes (Is) of each incubation exposed (min; t) to 1 g/l nicotine or not was fitted to the logistic equation Is = K/(1 + exp(a + bt)], where K = 1 due to Is ≤ 1, a is the intercept for the fitted curve, and b is the increasing rate of cellular viability under the stress. The equation was used to depict the viability of strain HF-1 in the presence or absence of nicotine. DPS software (Tang and Feng 2002) was used in all the analyses.
Native PAGE and Staining
Growth Curves of Strain HF-1
Effect of Different Concentrations of Nicotine on SOD Activity
Effect of Different Concentrations of Nicotine on CAT Activity
As expressed in Fig. 2b, CAT activities in treated growing cells were almost the same as that of control cells in the absence of nicotine treatment. However, compared to control cells, CAT activities in grown cells treated with 1.0, 1.3, and 2.0 g/l nicotine declined substantially (p < 0.01), suggesting the damage to bacterial cells caused by nicotine or its metabolites.
Effect of Different Concentrations of Nicotine on ATPase Activity
The variation of ATPase activities in growing and grown cells had great comparability with that of CAT activities, as shown in Fig. 2c. ATPase activities in grown Pseudomonas sp. HF-1 cells were inhibited significantly by all doses of nicotine (p < 0.01) and were reduced in a concentration-dependent manner, suggesting that the higher the concentration of nicotine, the stronger the inhibition. In contrast to grown cells, growing cells showed no difference in ATPase activity between all nicotine-treated cells and control cells and no obvious diversity of ATPase activity was observed between various treatments.
Response of SOD in Different Bacteria
Response of CAT in Different Bacteria
Compared to comparative strains, CAT activities in growing nicotine-degrading strain HF-1 enhanced notably after nicotine exposure, a 2.6-fold increase over that of the untreated cells (Fig. 3b). On the contrary, alteration of the relative CAT activities in growing non-nicotine-degrading strains was mild and the activities were almost identical to that of control cells not treated with nicotine. It was implied that CAT might occupy a crucial position in the resistance to the oxidant in Pseudomonas sp., whereas it has a limited role in the defense against nicotine in E.coli K12 and B. sutilis B19. In addition, the change of relative activities of CAT in the stationary-phase strain HF-1 was milder than that of exponential-phase cells. There was a weak stimulation in grown B. subtilis B19 after being exposed to nicotine for 4 h.
Response of ATPase in Different Bacteria
The relative ATPase activity markedly increased in growing E. coli after nicotine exposure and then declined fleetingly to a normal level upon further exposure (Fig. 3c). Furthermore, the ATPase activity in growing B. subtilis was apparently not stimulated until after being treated with nicotine for almost 10 h. In contrast to non-nicotine-degrading bacteria, total ATPase activities in growing HF-1 were much less than in the control. The relative activities in three stationary-phase cells were stimulated after the accession of nicotine and the maximal response to nicotine stress in grown strain HF-1 was attained after the cells had been exposed to nicotine for 4 h, longer than that of the comparative strains.
Activity Staining Profiles of SOD
Currently, there is considerable interest in free-radical-mediated damage in biological systems following nicotine exposure. However, these studies focused mainly on damages to mammiferous cells (Elkins et al. 1999; Kane et al. 2004; Diaz et al. 2000). In actuality, microorganisms frequently are exposed to this stress factor caused by tobacco planting, tobacco production, and the application of neonicotinoid insecticides. The ATPase, CAT, and SOD activities of nicotine-degrading bacterium Pseudomonas sp. HF-1 and two model strains treated with nicotine were detected and quantified in this study.
Oxidative stress arises during metabolism of naphthalene and other substrates by Pseudomonas sp. strain As1 and the stress can be reduced by antioxidant enzymes (Kang et al. 2007), as nicotine-degrading strain Pseudomonas sp. HF-1 did. Both SOD and CAT have been confirmed in their protection of host organisms from oxidant damage (Suntres 2002), but the protective function of these antioxidant enzymes is variable. Numerous studies have been carried out to research factors that affect SOD and CAT activities in microorganisms, and varied, even contradictory, results have been deduced. In E. coli, the SoxR regulon orchestrates genes for defense against certain types of oxidative stress through the SoxR-regulated synthesis of the SoxS transcription activator (Park et al. 2006). In this study, SOD plays a more important role in resistance to nicotine stress in E. coli than it does in CAT. However, the genetic responses to superoxide stress in Pseudomonas differ markedly from those seen in E. coli (Park et al. 2006), and CAT is important to Pseudomonas cells for acquired antioxidant resistance (Elkins et al. 1999). The results of this research indicate that multiple antioxidative enzymes are involved in antagonizing oxidative stress caused by nicotine in Pseudomonas sp. HF-1, and the contribution of a particular antioxidant enzyme for the protection from nicotine stress varies with the growth phase involved. Pseudomonas species have more complex regulatory systems to respond to nicotine stress than the E. coli system (Venturi 2003; Zheng et al. 2001).
During the past few years, concerns for the safety of our environment have increased enormously. A great number of different short-term bioassays based on bacteria have been proposed (Lampinent et al. 1995). The same chemical compound can result in a distinct response in Gram-positive and Gram-negative bacteria and the complex mechanism is still not very clear (Buurman et al. 2006). As can been seen, the antioxidant enzyme levels differ greatly between Gram-negative and Gram-positive strains. Gram-positive strain B. subtilis B19 appeared to be almost unresponsive to nicotine exposure, whereas Gram-negative bacteria E. coli K12 and Pseudomonas sp. HF-1 showed higher sensitivity to nicotine stress. Gram-negative bacteria are the more suitable organisms for studies concerning the action of environmental nicotine pollution.
Furthermore, it is well known that ATP is indispensable for the growth of organisms, and ATPase plays an important role in many intracellular physiological functions, which can be considered to be a sensitive indicator of toxicity (Yadwad et al. 1990). Significant inhibition of ATPase activity after nicotine exposure suggests that nicotine probably affected the metabolism of ATP to some extent.
Different from the fluctuation of specific SOD activities, the isoforms of SOD in B. subtilis B19 and strain HF-1 were steady except for staining intensity. However, the pattern of SOD in E. coli K12 altered significantly and the new isozyme band was exhibited. It is implied that bacteria act against oxidative stress not only by changing SOD activity but also by altering isoforms of SOD. This is an exciting event that can lead to the possible use of SOD, CAT, or ATPase as a bio-indicator for nicotine contamination.
This work was supported by grants from the National Natural Science Foundation of China (No.30570053, No.40501037), the Research Fund of Science and Technology Bureau of Zhejiang Province (No. 2008C23088), and the National Key Technologies Research and Development Program of China during the 11th Five-Year Plan Period (No. 2006BAJ08B01).