FLUORIDE 31 (2),
1998, pp 81 - 88		 International Society for Fluoride Research Table of Contents
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Research Report
EFFECT OF FLUORIDE ON SUPEROXIDE DISMUTASE (SOD)
ACTIVITY IN GERMINATING MUNG BEAN SEEDLINGS

L G Wilde* and M Yu†
Bellingham, Washington, USA

SUMMARY: The influence of sodium fluoride (NaF) on superoxide dismutase (SOD) in mitochondria from germinating mung bean seedlings was studied. Mitochondrial preparations were obtained from seedlings treated daily with 0 (control), 0.1, 0.2, 1, and 5 mM NaF for 72 hours. NaF at 0.1 mM caused a small but significant increase in SOD activity, whereas 1 and 5 mM NaF decreased the enzyme activity by 10 and 40%, respectively.

Key words: Mung bean (Vigna radiata), Seed germination, Sodium fluoride, Superoxide dismutase (SOD).

*Present address: 1124 Sudden Valley, Bellingham, WA 98226 USA. Correspondence to L G Wilde.
†Center for Environmental Science, Western Washington University, Bellingham, WA 98225-9181 USA.

INTRODUCTION
Fluoride has long been known as a potent metabolic inhibitor. Thus, fluoride can interfere with the metabolism of proteins, lipids, and carbohydrates.1-4 Although the mechanism involved in the inhibition is not completely understood, fluoride often inhibits enzymes that require such cofactors as Ca2+, Mg2+ , and Mn2+ ions. For example, NaF inhibited amylase5 and invertase6 in germinating mung bean (Vigna radiata) seeds. The inhibition is attributed, in part, to removal of the cofactor Ca2+.5,6 Changes in enzyme activity and intermediary metabolism caused by chronic fluoride exposure may lead to altered growth, development, and reproduction of the organism.

Among the many biochemical effects of fluoride, one that has attracted much recent attention is the generation of superoxide free radicals (O2–·). 7 Superoxide free radical (O2–·) is produced from O2 by both natural and anthropogenic processes. It is produced naturally during mitochondrial respiration, upon exposure to UV-B radiation, and during an immune response by phagocytozing cells.8-10 The anthropogenic processes of O2–· production are caused mainly by the action of various environmental pollutants such as NO2, CN–, and the herbicides paraquat and nitrofen.11-15

Superoxide free radical is both an oxidant and a reductant and has the potential to cause adverse effects on biomolecules. For example, it can damage membrane lipids through lipid peroxidation and cause enzyme inactivation and DNA strand breakage.16-18

Living systems have evolved an intracellular enzymatic defense system to protect themselves against O2–·. Superoxide dismutase (EC 1.15.1.1) (SOD) is an enzyme responsible for the breakdown of O2–·. It is a metalloprotein and catalyzes the dismutation of O2–· to O2 and H 2O2.19 By altering the concentration of O2–·, SOD helps prevent both direct toxicity from O2–· and secondary toxicity from ·OH and H2O2.

A large volume of literature exists describing various physiological and biochemical effects of fluoride on higher plants. Observed symptoms in plants include depressed growth and development, chlorosis, decreased photosynthetic activity, necrosis, abscission of leaves, flowers, or fruits, impaired cone and seed production, and necrosis. 20 Fluoride has been shown to inhibit the activity of SOD. For example, in vitro studies with E. coli exposed to 30 mM fluoride showed a 50% reduction in SOD activity.21 However, no information is available demonstrating in vivo inhibition of SOD activity by fluoride. We reported that exposure to 1.0 mM NaF inhibited mung bean germination, as manifested by decreased root elongation, altered tissue fatty acids1 and soluble sugar composition.6 In this communication, we report identification of SOD activity in mitochondrial preparations from mung bean seedlings and that NaF inhibited the enzyme in vivo.

MATERIALS AND METHODS
Seed germination and fluoride treatment: Mung bean seeds were germinated in an incubator at 25°C (±0.5°C). One-day-old seedlings were treated daily with 0 (control), 0.1, 0.2, 0.5, 1.0, or 5.0 mM NaF for 72 hours.

Preparation of crude extract: Crude extracts were obtained from whole seedlings or from the cotyledons, hypocotyls, and roots as needed. Extracts were prepared using the methods of Giannopolitis and Ries21 with a minor modification. Mitochondrial fractions were prepared by differential centrifugation and the precipitated mitochondria were homogenized using a cold mortar and pestle, resuspended in 0.05 M KH2PO4-NaOH buffer (pH 7.8), and used in SOD assay.

Assay of SOD activity: The xanthine/xanthine oxidase assay used was a compilation of the methods from various sources.19,23,24 Xanthine oxidase catalyzes the oxidation of xanthine to uric acid and in the process generates O2– (Figure 1). The O2–· production is coupled to the reduction of cytochrome C (a colorimetric reaction), which is followed spectrophotometrically, allowing for quantitative measurement. The SOD containing preparation added to the assay mixture will convert the superoxide free radical to H2O2 and O2, therefore slowing the rate of cytochrome C reduction. During the assay, the absorbance was determined in a sprectrophotometer (IBM UV-Vis 9420) fitted with a Haake Temperature Regulator maintained at 29°C (±1°C). Additional data were obtained using an Hewlett Packard HP8452A Diode Array spectrophotometer using HP Kinetics software and regulated by an IBM 9550 Heating Cooling Fluid Circulator.

The assay mixture consisted of 2.90 mL solution (Solution A) containing 6.2 m g xanthine and 65.4 m g cytochrome C in 0.05 M KH2PO4-NaOH buffer (pH 7.8) and 50 µL enzyme. Prior to addition of the enzyme, the absorbance of Solution A at 550 nm was followed for 2.0 minutes at a 30-second interval to establish a baseline slope. The enzyme extract (50 µL) was then added to Solution A and absorbance measurement was continued every 30 seconds for another 4.0 minutes. The resultant slope from 2.5 minutes to 5.5 minutes was then compared to the slope of the first 2.0 minutes to determine if the test solution could reduce the rate of cytochrome C reduction (Absorbance at 550 nm [A550] increase). Each sample was assayed at least twice, unless otherwise noted.

One unit of SOD activity was previously defined as a 50% decrease in the rate of cytochrome C reduction. Ideally, the initial rate of cytochrome C reduction before SOD addition was a change of 0.025 absorbance units per minute at 550 nm. To calculate the units of SOD activity in the assayed fraction, the rate of A550 increase from 2.5 to 5.5 minutes was divided by the initial rate from 0 to 2.0 minutes. This represents the percent difference in rate. This was subtracted from 100% to get the percent decrease in A550 increase. The resultant percent was then divided by 50% to normalize to units of SOD activity. These calculated units of SOD activity were then normalized for protein content by dividing by mg protein in the 50 µL sample assayed. Protein content of the sample was determined according to the method of Lowry et al.25 using bovine serum albumin (BSA) as a standard.

Statistics: Each experiment was repeated at least three times with new groups of seedlings. Two or three subsamples were also run on each experimental unit to observe the extent of assay variability. Wilk-Shapiro tests were conducted to confirm that data fit a normal distribution. Equal variance within the data was monitored using Bartlett's test of equal variances. Data that was not normally distributed or that did not have equal variance was log transformed. Randomized complete block style ANOVA tests were conducted, except where noted, with a p< 0.05 as the decision to reject or not reject the null hypothesis in each experiment. Asterisks on graphs denote statistical significance.

RESULTS AND DISCUSSION
Figure 2 shows the effect of varying in vitro pH (5.7, 6.4, 7.0, 7.4, 7.8, and 8.0) on mitochondrial SOD activity. No significant differences in the enzyme activity were observed within the pH range tested. However, the activity peaked at pH 7.8 and then sharply decreased. It appears that the optimum pH for the enzyme activity is at 7.8. This coincides with the optimum pH for SOD activity in peas and corn preparations reported by Giannopolitis and Ries.26

The effect of in vitro temperature changes on mung bean mitochondrial SOD activity was tested at 27, 30, 35, and 37°C. As shown in Figure 3, the enzyme activity increased with increase in temperature up to 37°C. Attempts to test the effect of temperature on mitochondrial SOD activity at 40°C were unsuccessful, because a baseline slope could not be established, possibly due to degradation of one or more components in the reaction mixture. It was apparent that even though activity could be maximized at 37°C, subsample variance also increased with increase in temperature. Thus, it can be concluded that using a lower assay temperature such as 30°C may provide more reproducible results.

The mitochondrial SOD activity increased with age of seedlings. The activity was very low in 2-day-old seedlings, but it increased nearly 8 and 20 times in 3-day-old and 4-day-old seedlings, respectively. SOD activity has been shown to increase with age of other test organisms. For example, total cytosolic and mitochondrial SOD activity in rat liver increased with age of the animal.27 The activity in pea plumule greening and oat seed germination also exhibited a similar trend.26 As growth continues during germination, the demand for respiration increases, so the number of mitochondria increases with age.

The enzyme activity differed within tissues, also. Based on the results of two separate experiments, it appears that the order of SOD activity in the tissues studied was hypocotyl>root>cotyledon (see Table).

TABLE. Mitochondrial SOD activity in cotyledons
from seedlings exposed to NaF
NaF concentration SOD activity (±SD)*
(mM) (units/mg protein)
0.0 21.1 (±7.9)
0.1 21.7 (±6.1)
0.5 16.7 (±3.9)
1.0 18.8 (±3.2)
  Values are the average of 3 determinations


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Figure 1. SOD assay reactions
Figure 2. Effect of pH on SOD activity
Figure 3. Effect of temperature on SOD activity
* indicates significantly different from each other (or from the control)
Figure 4. Effect of in vivo fluoride exposure on SOD activity in whole mung bean seedlings
* indicates significantly different from each other (or from the control)

Experiments were conducted to study the effect of in vivo fluoride exposure on SOD activity. For this purpose, seedlings exposed to varying concentrations of NaF for 72 hours were used to prepare the mitochondrial fraction. The results from experiments on mitochondrial preparations from whole seedlings are shown in Figure 4. A significant increase (10%) in enzyme activity occurred in seedlings exposed to 0.1 mM NaF. The activity was depressed steadily at 0.5, 1, and 5 mM NaF. In a separate set of experiments, the mitochondrial fraction was obtained from the cotyledons. The trend exhibited in these two sets of experiments was generally similar to each other. A slight increase in SOD activity in the 0.1 mM NaF group, and decreases in tissues treated with higher concentrations of NaF (see Table).

It is clear from the experimental results that treatment with NaF in vivo altered mitochondrial SOD activity in mung bean seedlings. It is interesting to note that NaF at low concentrations enhanced SOD activity, but depressed it at high concentrations (Figure 4). Although the mechanism involved in these changes is not clear, a similar trend has been observed in many toxicological studies.28,29 The slight increase in SOD activity seen in seedlings treated with 0.1 mM NaF may be partly due to an increased metabolic activity or an increased SOD biosynthesis induced by fluoride exposure. Reiss et al30 reported that, in rat brain and heart, inactivated SOD due to aging was replaced by new synthesis to maintain constant levels of the enzyme.

Acknowledgment: We thank the Bureau for Faculty Research and Huxley College, Western Washington University, for financial support. We also thank Terry Meredith, Clint Burgess, and Dr Don Williams for technical assistance.


REFERENCES
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FLUORIDE 31 (2),
1998, pp 81 - 88		International Society for Fluoride Research
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