FLUORIDE 28(4),
1995, pp 193-200		International Society for Fluoride Research
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Research Report

CHANGES OF THE HUMAN ERYTHROCYTE MEMBRANE
PROTEIN SH BINDING SITE PROPERTY WITH EXPOSURE
TO FLUORIDE AND THREE STRONG MUTAGENS

YY Wang, XJ Li and WJ Xin,   Beijing, China

SUMMARY: The effects of three strong mutagens (potassium bichromate, mitomycin C, and colchicine) and fluoride on the human erythrocyte membrane protein SH binding site property have been studied by using the maleimide nitroxide-ESR technique. The results indicate that in singular and combined treatments with mutagens, the ratio of weakly to strongly immobilized components is reduced, so that the conformation of the weakly immobilized component protein is altered. It is possible that the inhibition in the cytogenetic response is induced by the interaction of fluoride with the other chemicals. There is a dose and temperature dependence of both the singular and the combined action of the mutagen on the membrane protein.
Key Words: Dose; Erythrocyte; Fluoride mutagenicity; Human; Membrane protein SH binding site property; Strong mutagens; Temperature.

Introduction
Fluoride (F–) is a weak mutagen but inhibits a cytogenetic response induced by strong mutagens such as potassium bichromate (Cr 6+), mitomycin C (Mit C), and colchicine (Colch) by affecting sister chromatid exchange, micronuclei and cell cycle.1-5 The inhibition of the cytogenetic response in the interaction of F– with Cr 6+, Mit C and Colch has been postulated to be in relation to the conformational changes of membrane lipids and proteins involved in transport processes and permeability of the cell membrane.6 Therefore, a better understanding of the behavior of fluoride on the cell membrane might help clarify the inhibition due to the interaction of F– and other mutagens. Spin label electron spin resonance (ESR) technique is a valuable tool for studying the basic characteristics of biomembrane preparations, including conformation, permeability and fluidity.7-9 Maleimide nitroxide used as a spin label can reveal more changes of the physical characteristics of cell membrane than stearic acid 10-11 and the ratio of weakly to strongly immobilized components (W/S) of the ESR spectra is very sensitive to the conformational changes of protein.11 There is evidence of a close relationship between membrane protein SH groups and permeability.12 The study of the inhibition by F– of the effects of Cr 6+, Mit C and Colch on the membrane is based on earlier measurements of the latter three chemicals performed by using maleimide nitroxide-ESR technique. In the present study a new area of F– research is investigated: the inhibition of the cytotoxic response induced by Cr 6+, Mit C and Colch, through interfering with the membrane protein SH binding site properties.

Materials and Methods
Erythrocytes: Healthy human erythrocyte suspensions (ACD-B), containing 1x10 9 cells/ml, were supplied from the blood centre of the Red Cross Society in Beijing.

Spin labels: 3-Maleimide-PROXYL I was purchased from Aldich Co., USA. 7.5 x 10 -4 mM maleimide solutions were prepared with isotonic Tris buffer.

Mutagens: Sodium fluoride and potassium bichromate AR were produced by BHC China. Mitomycin C injection was purchased from Kyowa, Japan. Colchicine was obtained from Fluk Co., USA.

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Procedure: Dilutions of an erythrocyte suspension containing 1x107 cells/ml were prepared in Tris-isotonic solution (1:100). The experiment was divided into two parts. In the singly treated group, Cr6+, Mit C and Colch were singly added to the diluted erythrocyte suspensions. In the combined treated group, F– with Cr6+, Mit C and Colch respectively was added to the erythrocyte dilutions. A control group without adding any chemicals was set. After shaking and incubating for 15 h at 37°C, the mutagen-exposed erythrocyte solutions were centrifuged at 1000 g for 8.0 min. The supernatants were then added to maleimide spin label solutions (final concentration was 1.5 x 10-4 M) to label the mutagen-treated red cells. All the samples were incubated for 2.0 h at 37°C and subsequently centrifuged at 1000 g. The residual cells were rinsed three times with isotonic solutions to make the supernatants free from any signals of free spin labels, so that the ESR spectra would come only from the spin labelled erythrocytes. The spin-labelled cells were then put into a quartz capillary for ESR measurement, as previously described.10-11

Results and Discussion
Figure 1. A-B shows the representative ESR spectra obtained from human intact erythrocyte membrane, labelled with maleimide nitroxide I, after the mutagen exposure. Analysis of these spectra indicated that 2A values calculated from ESR spectra are identical: 66.7 ± 1.85G (mean ± standard error, SE) with a slight fluctuation of their peak amplitudes due to mutagen presence. The alterations of the signal amplitude are the result of both weakly and strongly reacting components, namely, the singular and combined treatments. It is possible that after exposure of cell membranes to any mutagen, the motions of the maleimide probes attached to immobilized binding sites do not encounter restrictions, and these probes do not move close enough to each other to result in dipolar broadening. It has been proven that the conformational changes of the membrane proteins could not be induced by exposure to the mutagen.

As shown in Table 1, the W/S value from human erythrocyte membrane exposed to Cr6+, Mit C and Colch and labelled by maleimide is basically less than that of the untreated control group. The W/S ratio of erythrocyte membrane treated by Cr6+ and Mit C decreases with the increasing dose. However, the W/S ratio with Colch exposure decreases at low concentration, but increases at high concentration, the increase starting at dose 7.5 µM.

When these results were compared to the effect of F– alone,10 the greatest reduction of the W/S ratio of the human erythrocyte membrane was found with Cr6+ and the least with Colch.

[figure not shown here]



Table 1. W/S ratio of the human erythrocyte membrane before and after the treatment with various doses of mutagens
Dose Cr6+ Dose Mit C Dose Colch
µM Mean ± SE µM Mean ± SE µM Mean ± SE
0 1.986 0.094 0 1.986 0.094 0 1.986 0.094
0.15 1.266 0.118 0.6 1.408 0.031 5.0 1.585 0.112
0.30 0.956 0.073 1.2 1.292 0.180 7.5 1.593 0.086
0.60 0.651 0.031 1.8 1.205 0.162 10.0 1.651 0.096
0.90 0.532 0.027 2.4 1.149 0.180 12.5 1.729 0.126

The results in Table 2 show that the W'/S' ratio, with the combined exposure of F– with Cr6+ Mit C and Colch, decreases with increasing F– dose. That is, deltaW'/S', the difference between W'/S' ratio of F– and Cr6+, Mit C and Colch, increases with the F– dose (Figure 2).

W"/S" ratio in Table 3 decreases with the increasing dose of Cr6+ and Mit C with the combined exposure of a given dose of F– (37.5 µM). Specifically, the deltaW"/S" ratio, the difference between W"/S" of F– and Cr6+ or Mit C, increases with the dose of Cr6+ or Mit C respectively. The comparison shows that the difference between the inhibitory strength of Cr6+ or Mit C due to F– is the same as the results in Table 2. However, W"/S' ratio of Colch decreases in low concentration and increases with higher concentrations when combined with a 37.5 µM F– exposure. Thus, W"/S" ratio exceeds W/S value of 37.5 µM F– exposure at the time Colch dose reaches 12.5µM, exhibiting the same response as Colch singularly treated erythrocyte membrane. As a result deltaW"/S" ratio, the difference between W"/S" ratio of F– and Colch exposure, becomes negative. That is, the point of deltaW"/S" lies in the fourth quadrant in Figure 3.


Table 2. W'/S' ratio when the various doses of F–
were combined with Cr6+, Mit C or Colch
Group F–(µM)
    0 5.0 12.5 25.0 37.5
Cr6+ Mean 0.532 0.530 0.479 0.477 0.467
(0.9 µM) ± SE 0.027 0.069 0.054 0.050 0.045
Mit C Mean 1.149 0.930 0.866 0.820 0.764
(0.4 µM) ± SE 0.180 0.042 0.047 0.040 0.018
Colch Mean 1.585 1.358 1.319 1.205 1.167
(5.0 µm) ± SE 0.112 0.005 0.051 0.021 0.035

[Figure not displayed here]
Figure 2. The dose curves in W'/S' of sodium fluoride
Cr6+    Mit C    Colch
W'/S' : W/S ratio obtained from combined treatment with a certain dose of Cr6+, Mit C or Colch with various doses of F–.
deltaW'/S' : The difference between W'/S' of F– and W'/S' of Cr6+, Mit C or Colch.

Analysis of the above data also suggests that there is a parallel relationship between the membrane protein SH binding site property and the dose of mutagens in the singular and combined treatments.

The results of reductions in the W"/S" ratio in singular treatment, with F–, Cr6+ and Mit C may occur through the same mechanism with the oxidation of protein SH groups on the human erythrocyte membrane to S-S linkages. As is well known, Cr6+ is a very strong oxidant.13


Table 3. W"/S" ratio when the various doses of
Cr6+, Mit C or Colch were combined with 37.5 µM F–
Cr6+ Dose (µM) 0 0.15 0.30 0.60 0.90
Mean 1.360 0.990 0.852 0.619 0.542
± SE 0.065 0.023 0.026 0.026 0.014
Mit C Dose (µM) 0 0.6 1.2 1.8 2.4
Mean 1.360 1.069 0.982 0.944 0.895
± SE 0.065 0.095 0.112 0.103 0.127
Colch Dose (µM) 0 5.0 7.5 10.0 12.5
Mean 1.360 1.281 1.311 1.334 1.373
± SE 0.065 0.111 0.059 0.062 0.063

[figure not included -- see subscription info for details on how to subscribe]
Figure 3. The dose curve in W"/S" of Cr6+, Mit C or Colch
Cr6+    Mit C    Colch
W"/S": W/S ratio obtained from combined treatment with 37.5 µM F– respectively with various doses of Cr6+, Mit C or Colch.
deltaW"/S": The difference between W"/S" of F– and W"/S" of Cr6+, Mit C or Colch.

Mit C contains a quinone group and can release an active oxygen free radical,14 and F– is strongly electronegative.15 Besides, analysis of ESR spectra reveals that the reductions of W/S ratio are not the results of damage to cell membranes (constant W while S value changes) but rather arise mainly from a decrease of the weakly immobilized component. The oxidations by the three mutagens of the SH-groups of the weakly immobilized component of the protein may be explained by the actions of the chemicals on the membrane surface only. That is, the contribution to the SH/S-S rate in the membrane depth is higher than that in the surface. In consequence, the conformational changes of membrane protein are induced by mutagens.

Colchicine is a mitotic poison distinguished from the other three mutagens as a chromosomal clastogen. Although Colch can cause the changes of the association and functioning of microtubules and microfilaments due to non-covalent binding with microtubule protein, its chemical properties are not yet fully understood.16 Colch appears to decrease the weakly immobilized component protein SH groups on erythrocyte membrane. The phenomenon whereby the W/S ratio is decreased and then increased in the course of Colch exposure, as seen in Figures 2 and 3, is not understood at present.

The results in Table 4 show that the W/S ratio of erythrocyte membrane, singly treated by Cr6+, Mit C and Colch and in the control, increases with the temperature between 0 and 50°C. A point of phase transition, in the course of the temperature changes, exists in each measurement group: 28.5°C in the control, 28.8°C in Cr6+, 25.5°C in Mit C, 24.0°C in Colch, and 22.5°C in F-10 (Figure 4).

The results in Table 5 indicate that the W/S ratio of erythrocyte membrane jointly treated by F– with Cr6+, Mit C and Colch increases with the temperature between 0 and 50°C. A point of phase transition exists in each group: 24.0°C in F–- Cr6+, 25.0°C in F– Mit C and 27.5°C in F–- Colch (Figure 5).

There is a temperature dependence of the W/S ratio in cell membrane treated by Cr6+, Mit C, Colch and F– singly, and by F– with the former three mutagens jointly. A transition temperature exists with each group. Compared with the control, the phase transition point in erythrocyte membrane exposed to F–, Mit C and Colch is decreased 6.0°C, 3.0°C, and 4.5°C, but with Cr6+ is increased 0.3°C, respectively, while with F–- Cr6+, F– - Mit C and F–- Colch the transition point is reduced 4.5°C, 3.5°C and 1.0°C, respectively. As compared with the corresponding mutagen, there is 4.8°C and 0.5°C decrease of Cr6+ and Mit C but 3.5°C increase of Colch.

The alteration of the phase transition point caused by F– of 6.0°C was greater than that caused by the other mutagens Mit C, Colch and Cr6+. This fact suggests that F– has a stronger effect on the membrane protein SH binding sites than the other three mutagens. When F– was combined with the other three mutagens the difference between the phase transition points was not enhanced as much as with F– alone.

Temperature appears to affect the membrane protein structure so that the W/S ratio increases with the temperature, inducing change in the membrane conformation. The result is that the W/S ratio of the treated group is less than that of the control, because the more the protein SH group of the weakly immobilized binding site is exposed with temperature change, the more the SH group is affected by the mutagen.

Table 4. Change of W/S ratio with temperature
in singular treatment with mutagens
Group Temperature 0°C 10°C 20°C 25°C 30°C 35°C 40°C 50°C
Control Mean W/S 0.697 0.918 1.136 1.257 1.356 1.520 1.639 1.944
(0.0µM) ± SE 0.012 0.010 0.014 0.008 0.014 0.026 0.024 0.067
Cr6+ Mean W/S 0.301 0.387 0.470 0.523 0.585 0.639 0.725 0.917
(0.9Mµ) ± SE 0.031 0.031 0.039 0.044 0.056 0.064 0.068 0.095
Mit C Mean W/S 0.475 0.639 0.762 0.826 0.921 1.003 1.100 1.318
(2.4µM) ± SE 0.013 0.025 0.024 0.021 0.012 0.018 0.011 0.014
Colch Mean W/S 0.689 0.902 1.128 1.230 1.363 1.501 1.630 1.907
(5.0µM) ± SE 0.017 0.022 0.005 0.012 0.033 0.031 1.027 0.042

Figures not featured here
Figure 4 (below left) Temperature curve of human erythrocyte membrane before and after singular treatment with mutagens.
Control    Cr6+    Mit C    Colch

Figure 5 (below right) Temperature curve of human erythrocyte membrane before and after combined treatment with mutagens. Cr6+    Mit C    Colch

Table 5. Change of W/S ratio with temperature
in combined treatment with mutagens
Group Temperature 0°C 10°C 20°C 25°C 30°C 35°C 40°C 50°C
F–- Cr6+ Mean W/S 0.283 0.364 0.448 0.500 0.556 0.619 0.703 0.876
(37.5-0.9µM) ± SE 0.017 0.023 0.027 0.028 0.029 0.042 0.046 0.041
F–- Mit C Mean W/S 0.429 0.584 0.734 0.779 0.889 0.991 1.069 1.330
(37.5-2.4µM) ± SE 0.062 0.072 0.106 0.110 0.118 0.145 0.131 0.178
F–- Colch Mean W/S 0.614 0.761 0.954 1.040 1.161 1.259 1.453 1.634
(37.5-5.0µM) ± SE 0.020 0.019 0.018 0.020 0.015 0.020 0.042 0.058

Conclusion
The results of the present study coupled with our previous work demonstrate that the fluidity and protein SH binding site property in human erythrocyte membrane are changed by the four mutagens fluoride, potassium bichromate, mitomycin C and colchicine. The conformations of the membrane lipids and proteins are also changed with resulting alterations in membrane function including permeability. When fluoride interacts with the other three mutagens the changes are inhibited. This inhibition results in a molecular response, possibly through the transmembrane protein of the erythrocyte membrane Band 3, which mediates anion exchange across the red cell membrane. Further exploration of the action of fluoride on the cell membrane may help ways to be found for protection against fluoride toxicity.

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Authors: YY Wang, XJ Li and WJ Xin
Beijing Municipal Research Institute of Environmental Protection,
Fu Wai Avenue, Beijing 100037, China.

FLUORIDE 28(4),
1995, pp 193-200		International Society for Fluoride Research
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