Lack of effect of a 60 Hz magnetic field on biomarkers of tumor promotion in the skin of SENCAR mice

John DiGiovanni4, Dennis A. Johnston, Tim Rupp, Lyle B. Sasser1, Larry E. Anderson1, James E. Morris1, Douglas L. Miller1, Robert Kavet2 and Earl F. Walborg, Jr3

University of Texas M.D.Anderson Cancer Center, Science Park–Research Division, PO Box 389, Smithville, TX 78957,
1 Battelle, Pacific Northwest Laboratories, PO Box 999, Richland, WA 99352,
2 EPRI, 3412 Hillview Avenue, Palo Alto, CA 94304 and
3 Dermigen Inc., PO Box 727, Smithville, TX 78957, USA


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
It has been proposed that extremely low frequency magnetic fields may enhance tumorigenesis through a co-promotional mechanism. This hypothesis has been further tested using the two-stage model of mouse skin carcinogenesis, i.e. 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced promotion of skin carcinogenesis in mice initiated by a single subcarcinogenic dose of 7,12-dimethylbenz-[a]anthracene. Experimentation utilized three different doses of TPA within its dose–response range (0.85, 1.70 or 3.40 nmol) and examined the following early biomarkers of tumor promotion after 1, 2 and 5 weeks of promotion: increases in epidermal thickness and the labeling index of epidermal cells, induction of epidermal ornithine decarboxylase activity and down-regulation of epidermal protein kinase C activity. Mice exposed to a 60 Hz magnetic field having a flux density of 2 mT for 6 h/day for 5 days/week were compared with mice exposed to an ambient magnetic field. Within the sensitivity limits of the biomarker methodology and the exposure parameters employed, no consistent, statistically significant effects indicative of promotion or co-promotion by the magnetic field were demonstrated.

Abbreviations: BrdUrd, 2-bromodeoxyuridine; DMBA, dimethylbenz[a]anthracene; ELF, extremely low frequency; ODC, ornithine decarboxylase; PKC, protein kinase C; TPA, 12-O-tetradecanoylphorbol-13-acetate.


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Although the scientific evidence has been deemed insufficient to support a definitive role of extremely low frequency (ELF) electromagnetic fields in the development of cancer (1,2), several exposure-associated observations have stimulated research on the possible cancer risk. One hypothesis proposes that exposure to a magnetic field exerts a co-promotional effect on tumor development (3). This hypothesis was tested by Stuchly et al. (3) using the SENCAR mouse skin model (4), where mice are initiated with a single subcarcinogenic dose of 7,12-dimethylbenz[a]anthracene (DMBA) and promoted by repetitive applications of 12-O-tetradecanoylphorbol-13-acetate (TPA). A co-promoter in this model is defined as an agent that possesses no direct tumor-promoting activity, but synergizes with a tumor promoter to enhance papilloma development. In the experiment performed by Stuchly et al. (3) one group of mice promoted with TPA (4.9 nmol/week for 23 weeks) was exposed to a 2 mT (60 Hz) magnetic field during tumor promotion and another group was not. The authors reported a statistically significant increase in tumor incidence and multiplicity at 16–18 weeks of promotion in the magnetic field-exposed group, but the effect became non-significant at 23 weeks of promotion. Subsequently, McLean et al. (5) reported the results of three replications of the same co-promotion experiment. At 23 weeks of TPA promotion, only one replication exhibited a statistically significant effect on tumor incidence and multiplicity; and the direction of that effect was opposite to that predicted for co-promotion.

Recently the same SENCAR mouse model was used to test for a co-promotional effect of a 2 mT (60 Hz) magnetic field on tumor development using three different TPA doses within its dose–response range (6). Evaluation of the effects of the magnetic field on tumor incidence and multiplicity revealed no statistically significant co-promotional effects within the sensitivity limits imposed by the animal model and the exposure parameters employed. Correlative assessment of magnetic field-induced co-promotion also included monitoring of early biomarkers of tumor-promoting activity associated with sustained, potentiated epidermal hyperplasia in mouse skin (4) and this assessment is reported herein. Epidermal hyperplasia-associated effects were monitored as an increase in epidermal thickness or the labeling [2-bromodeoxyuridine (BrdUrd)] index of epidermal cells (7), by the induction of epidermal ornithine decarboxylase (ODC) activity (810) and by the down-regulation of epidermal protein kinase C (PKC) activity (11).


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Experimental animal model, design of the magnetic field exposure facility and parameters of the exposure
These aspects of the experimentation have been reported previously (6). The experimentation described herein was performed using female SENCAR mice incorporated within the protocols previously described (6). The term `nulled field' used herein refers to nulling of the imposed magnetic field, but not the geomagnetic field. The pilot experiment included several TPA-treated groups (i.e. 0.21, 0.42, and 6.8 nmol TPA, administered in 200 µl acetone, twice per week) not mentioned in the previous report.

Experimental design
Pilot experiment.
A pilot experiment was performed to select appropriate doses of TPA to be used in the primary experiment and to identify any environmental effects on biomarkers of skin tumor promotion that could be attributed to housing within an energized exposure unit in which the magnetic field had been nulled. Comparisons were made between mice housed in a nulled field within the magnetic field exposure facility and mice housed in a separate room within the magnetic field exposure facility and subject only to the ambient geomagnetic field and minimal stray fields present in the facility (6). The following chemical treatment groups (32 mice/group) were employed: Group 1, acetone vehicle control; Groups 2–7, 0.21, 0.42, 0.85, 1.7, 3.4 and 6.8 nmol TPA, respectively (treatment groups described in Table IGo). All mice were initiated by a single topical application of 10 nmol DMBA/200 µl acetone. Sixteen mice in each chemical treatment group were exposed to the nulled or ambient magnetic field. Thirty minutes prior to killing, mice received i.p. injections containing 100 µg BrdUrd/g body wt. BrdUrd (Sigma, St Louis, MO) was dissolved in sterile phosphate-buffered saline 1 h before dosing and administered in 0.1–0.2 ml. At 6 or 48 h after the fourth dose of TPA (i.e. after 2 weeks of TPA-induced promotion), eight mice from each treatment (TPA dose/magnetic field exposure) group were killed and skin specimens excised and processed for evaluation of biomarkers of tumor promotion.


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Table I. Treatment-related effects on biomarkers of tumor promotion: pilot experiment
 
Primary experiment.
The primary experiment utilized three different doses of TPA (0.85, 1.7 and 3.4 nmol/200 µl acetone, twice per week) that had been selected on the basis of the pilot experiment that included the assessment of tumor development as previously reported (6) and the assessment of the early biomarkers of tumor promotion described herein. The eight treatment groups (24 mice/group) are described in Table IIGo.


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Table II. Effect of magnetic field (2 mT) exposure on biomarkers of tumor promotion: primary experiment
 
Thirty minutes prior to killing, mice received i.p. injections of BrdUrd as described above. Four mice from each treatment group were killed at 6 or 48 h after application of the second, fourth and tenth doses of TPA (i.e. after 1, 2 or 5 weeks of TPA-induced promotion) and skin specimens excised and processed for evaluation of the early biomarkers of tumor promotion.

Collection of skin specimens
Following death, mice were shaved to remove hair from the treated dorsoscapular area of the back and, immediately thereafter, a depilatory (Nair) was applied. The backs of the mice were kept on ice until its application. After the depilatory had been on the skin for 1–2 min, the backs of the mice were gently scrubbed under cold tap water. An elliptical section of dosed skin (~2 cm widex3.5 cm long, with its midline axis above the spinal column) was excised and kept on ice until further processing. The excised skin was cut perpendicular to its long axis to yield equal halves. One specimen (~1x1.75 cm, cut from the anterior half and having its lateral edges ~0.5 cm from the midline) was fixed in buffered formalin and reserved for histomorphic and immunohistochemical evaluation. The posterior portion of the excised skin was divided at the midline to yield two specimens (~1x1.75 cm each). One specimen was processed for the assay of epidermal ODC activity and the other for the assay of epidermal PKC activity. The formalin-fixed specimens were embedded in paraffin and processed for histomorphic and immunohistochemical evaluation. Care was taken to ensure that tissue sections were cut perpendicular to the plane of the skin.

Evaluation of early biomarkers of tumor promotion
Measurement of epidermal thickness.
Measurements of the thickness (µm) of the non-cornified interfollicular epidermis were performed on hematoxylin and eosin stained histological sections (10 random 40x fields). The methodology followed essentially that described by Naito et al. (7). A mean epidermal thickness was calculated for each animal. A mean of means was then calculated for each treatment/sacrifice group.

Labeling index of epidermal cells using incorporation of BrdUrd.
BrdUrd-labeled epidermal cells were detected in histological sections of mouse skin using essentially the immunohistochemical methodology of Sugihara et al. (12). Random interfollicular fields were selected for scoring of BrdUrd-labeled nuclei by light microscopic examination. A total of 500 basal cells (labeled and unlabeled) adjacent to the basement membrane were evaluated and the labeling index expressed as percent labeled cells. A mean labeling index was calculated for each animal. A mean of means was then calculated for each treatment/exposure time group.

Induction of epidermal ODC activity.
Epidermal ODC activity was measured essentially according to the methodologies described by O'Brien and Diamond (13) and Kruszewski and DiGiovanni (10), using the liberation of 14CO2 from [14C]ornithine. Enzyme activity is expressed as nmol CO2 liberated/mg protein/h. Unfixed skin specimens reserved for this assay were placed on a cold glass plate and the epidermis scraped from the underlying dermis. The scrapings from all mice in each treatment/sacrifice group were pooled and homogenized (4°C) in 2 ml of the buffer described by Kruszewski and DiGiovanni (10), using a Polytron homogenizer (2x15 s). The homogenate was centrifuged at 27 000 g for 30 min at 4°C. The supernatants were stored at –70°C until use in the assay. Each supernatant was assayed using replicate 100 µl aliquots. The protein content of the supernatants was determined using the Bio-Rad Protein Assay kit (Bio-Rad, Richmond, CA).

Down-regulation of epidermal PKC activity.
Epidermal PKC activity was measured by the methodology described by Hirabayashi et al. (11). Unfixed skin specimens reserved for this assay were placed on a cold glass plate and the epidermis scraped from the underlying dermis. The scrapings from all mice in each treatment/sacrifice group were pooled and homogenized (4°C) in 2 ml of the buffer described by Hirabayashi et al. (11), using a Polytron homogenizer (2x10 s). A cytosolic fraction of the homogenate, partially purified by chromatography on small columns of DEAE–cellulose, was assayed for PKC activity as measured by the incorporation of 32P from [{gamma}-32P]ATP into H1 histone. Specific activity was expressed as c.p.m./µg protein eluted from the DEAE–cellulose. Protein content of the eluate from the DEAE–cellulose column was determined using the Bio-Rad Protein Assay kit. Down-regulation of PKC was also expressed as a percentage of the appropriate control, i.e. acetone-treated skin.

Statistical methods
Data on the biomarkers of tumor promotion were evaluated statistically using the analysis of variance (ANOVA) (14). In the pilot experiment two-factor (TPA treatment and room location: ambient or null field) analyses were performed on data obtained at the indicated exposure times. Since assay of epidermal ODC and PKC activities required the pooling of epidermal cells from each treatment group, there was no within statistical cell error (i.e. no replications). In these cases the interaction term was used as the error term in the statistical analysis. Measurements of epidermal thickness and the labeling index of epidermal cells were performed on skin specimens from each mouse in the treatment group; consequently such data were processed normally.

In the primary experiment, data were collected after three different durations of TPA promotion (i.e. 1, 2 and 5 weeks); consequently three-factor (TPA treatment, magnetic field exposure and week of promotion) analysis could be used. When there was no week of promotion effect, data from all three weeks were pooled, thereby providing a within statistical cell error for epidermal ODC and PKC activities. This, in turn, increased the error degrees of freedom and the precision of the estimation of random error.

The data were analyzed using SPSS (SPSS, Chicago, IL) or STATISTICA (StatSoft, Tulsa, OK). Statistical significance was set at P < 0.05.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Some data mentioned in the results are not included in Tables I and IIGoGo, but may be accessed in a published report of the Electric Power Research Institute (15).

Pilot experiment
This experiment was designed to detect differential effects on early biomarkers of tumor promotion attributable to housing within an energized exposure unit in which the magnetic field had been nulled. Mice exposed to the nulled magnetic field within the exposure facility were compared with mice housed in a comparable system located in a separate room and exposed only to the ambient geomagnetic field and minimal stray fields present in the facility (6). The effects of TPA on the biomarkers of tumor promotion in mice housed in the two rooms are shown in Table IGo.

Epidermal thickness was significantly increased by TPA treatment in a dose-dependent manner at doses >=0.42 nmol at both 6 and 48 h after a 2 week TPA treatment. Since a maximal epidermal response has been demonstrated to occur at ~48 h post-final dosing using this treatment regimen (16), only those data are shown in Table IGo. The greatest increase in epidermal thickness was observed at 6.8 nmol TPA, i.e. the highest dose tested. Two-way ANOVA (factors: TPA treatment and room location) of TPA-induced increases in epidermal thickness revealed no significant location-associated differences at either killing time. When the data from the 48 h experiment were analyzed, TPA treatment effects were highly significant (P = 0.001), but differences attributable to room location were not (P = 0.10). The TPA treatment differences were indicative of a non-linear dose–response effect.

The highest TPA-induced increases in the labeling index of epidermal cells occurred at 48 h post-final dosing, an observation again consistent with previously published data (7). At 48 h post-final dosing the labeling index exhibited a dose–response effect similar to that observed for epidermal thickness, with the highest indices observed at a dose of 6.8 nmol TPA. Two-way ANOVA revealed a highly significant TPA treatment effect (P < 0.001) and a significant room location effect (P = 0.038). The statistically significant difference attributable to room location disappeared if the aberrantly high labeling index exhibited by the vehicle control exposed to the ambient field was excluded (P = 0.11). The TPA treatment differences were again indicative of a non-linear dose–response effect.

At 6 h post-final dosing epidermal ODC activity increased as a function of TPA dose and the induction was maximal or near maximal at 3.4 nmol TPA. Consistent with published observations (10), epidermal ODC activity was substantially reduced by 48 h post-final dosing (data not shown). Two-way ANOVA was performed only on ODC activities of skin specimens obtained at 6 h post-final dosing, i.e. the approximate time of maximal enzyme induction (13,15). Statistical analysis of these data revealed significant TPA treatment effects (P < 0.001), but no room location effects (P = 0.43).

In general, PKC was down-regulated by TPA in a dose-dependent manner at 6 h (data not shown) or 48 h post-final dosing, with maximal or near maximal down-regulation occurring at TPA doses >=3.4 nmol. PKC data were analyzed by two-factor ANOVA, using the raw data (c.p.m./µg protein) at 6 or 48 h post-final dosing. Based on this analysis, TPA treatments were significantly different (P < 0.001), while the room locations were not (P > 0.13).

Primary experiment
The primary experiment examined the effect of a 2 mT (60 Hz) magnetic field on early biomarkers of TPA-induced tumor promotion. Based on the results of the pilot experiment, three doses of TPA within its dose–response range were employed, i.e. 0.85, 1.70 and 3.40 nmol. Comparisons were made between mice exposed to the 2 mT or ambient magnetic fields. The biomarkers were evaluated after 1, 2 and 5 weeks of TPA-induced promotion. Treatment-related effects on the biomarkers of tumor promotion are shown in Table IIGo.

For epidermal thickness, only data at 48 h post-final dosing were collected. A TPA dose-dependent increase in epidermal thickness was sustained throughout the first 5 weeks of tumor promotion in mice exposed either to the 2 mT or ambient magnetic field. When analyzed using a three-factor ANOVA (factors: TPA treatment, magnetic field exposure and week of promotion), no statistically significant effects on epidermal thickness could be attributed to the imposed magnetic field. As expected, a highly significant TPA treatment effect (P < 0.0001) was observed. There was no week of promotion (P = 0.086) or magnetic field exposure (P = 0.62) effect. None of the three possible two-factor interactions (i.e. TPA treatment/week of promotion, TPA treatment/magnetic field exposure or week of promotion/magnetic field exposure) were significant (P > 0.09).

Repetitive doses of TPA produced a 5- or 6-fold maximal increase in the labeling index of epidermal cells of mice exposed to a 2 mT or ambient field, respectively. When analyzed using the same three-factor ANOVA described above, no consistent statistically significant effects on the labeling index of epidermal cells could be attributed to the imposed magnetic field. As expected, a highly significant TPA treatment effect (P < 0.0001) was observed. There was a significant week of promotion effect (P = 0.026); but no significant effect of magnetic field exposure (P = 0.30). Of the three possible two-factor interactions (above), the interaction of TPA treatment with 1 week of promotion was significant (P = 0.0007). The interactions of magnetic field exposure with TPA treatment (P = 0.087) and 1 week of promotion (P = 0.14) were not significant.

Consistent with published reports demonstrating that TPA produces a sharp maximal induction of epidermal ODC activity at ~6 h post-final dosing (10,17), all three doses of TPA produced substantial induction of ODC activity within that time frame. At 48 h post-final dosing with TPA, epidermal ODC activities had fallen substantially to basal or near basal levels (data not shown). No statistically significant differences in the induction of epidermal ODC activity could be attributed to the imposed magnetic field. When analyzed using the three-factor analysis described above, there was, as expected, a highly significant TPA treatment effect (P = 0.0002). There was a week of promotion effect (P = 0.0002), but no magnetic field exposure effect (P = 0.58). The interaction of TPA treatment with week of promotion (P = 0.013) was significant;however, the interactions of TPA treatment (P = 0.37) and week of promotion (P = 0.60) with magnetic field exposure were not significant.

All three doses of TPA produced substantial downregulation of epidermal PKC activity and the response was dose-dependent. Although a statistically significant effect of the magnetic field on the down-regulation of epidermal PKC activity was observed using the data from the 6 h experiment, the effect was not observed in skin specimens collected from the 48 h experiment. The PKC data, expressed as c.p.m./µg protein, were analyzed by three-factor ANOVA. Using the raw data from the 6 h experiment, there was, as expected, a highly significant TPA treatment effect (P < 0.0009). There were also significant week of promotion (P = 0.0015) and magnetic field exposure (P = 0.006) effects. The two-factor interactions of TPA treatment with week of promotion (P = 0.007) and of TPA treatment with magnetic field exposure (P = 0.0059) were significant. The interaction of week of exposure with magnetic field exposure (P = 0.32) was not significant. The groups exposed to the 2 mT magnetic field had a greater overall mean down-regulation (3607 c.p.m./µg protein) than those exposed to the ambient field (5825 c.p.m./µg protein). Visual scanning of the data from the 6 h experiment showed that the week 2 values in general and the controls in particular were far below the levels observed at weeks 1 and 5. To ascertain if the significance and interactions may have resulted from the week 2 values, the three-factor analysis was re-run without week 2. The two-factor interactions were no longer significant (P > 0.08), while the TPA treatment effect remained significant (P = 0.0001). Neither the effect of magnetic field exposure (P = 0.11) nor week of promotion (P = 0.83) was significant. Using the raw data from the 48 h experiment, there was, as expected, a highly significant TPA treatment effect (P < 0.0001) and a significant week of promotion effect (P < 0.0001), but the magnetic field exposure effect was not significant (P = 0.17). The two-factor interaction of TPA treatment with week of promotion was significant (P = 0.0001); however, interactions of the magnetic field with TPA treatment (P = 0.078) and week of promotion (P = 0.35) were not significant.

To ascertain if the imposed magnetic field exhibited direct effects on DMBA-initiated mouse skin, statistical analyses of differences between Groups 1 and 5 (primary experiment) were performed using two-factor ANOVA. No significant effects of magnetic field exposure on epidermal thickness (48 h experiment; P = 0.99), labeling index of epidermal cells (48 h experiment; P = 0.65), epidermal ODC activity (6 h experiment; P = 0.10) or epidermal PKC activity (6 and 48 h experiment; P = 0.07 and 0.74, respectively) were observed.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The investigation described herein on the effects of exposure to a 2 mT magnetic field on early biomarkers of epidermal hyperplasia in SENCAR mice complements earlier observations (6) on the development of skin tumors in the same experimental model. The earlier study (6) did not detect any statistically significant effects of magnetic field exposure on the incidence or multiplicity of DMBA/TPA-induced skin tumors.

The pilot experiment described herein provided valuable information relevant to the execution of the primary experiment. First, it was demonstrated that TPA doses within the range evaluated in the pilot experiment were appropriate for the detection of effects on early biomarkers of tumor promotion, monitored by increases in epidermal thickness or the labeling index of epidermal cells, by the induction of epidermal ODC activity or by the down-regulation of epidermal PKC activity. Three TPA doses (0.85, 1.70 and 3.40 nmol), spanning the dose–response range, were selected for evaluation in the primary experiment. These same three doses were also utilized to evaluate the effect of a 2 mT (60 Hz) magnetic field on tumor development reported earlier (6). Second, comparisons between mice housed in the magnetic field exposure facility and exposed to a nulled magnetic field and similarly housed mice exposed only to the ambient geomagnetic field and minimal stray fields present in the facility revealed no significant location-related effects on early biomarkers of mouse skin tumor promotion by TPA. This observation demonstrated that mice housed in a room separate from the activated magnetic field exposure system can serve as an appropriate control for mice exposed to the 2 mT magnetic field, and such a control was used in the primary experiment.

The primary experiment demonstrated that the 2 mT magnetic field did not exert any consistent, statistically significant co-promotional effects on DMBA/TPA-treated SENCAR mice, monitored as effects on the four early biomarkers of tumor promotion in mouse skin. A statistically significant effect of magnetic field exposure on the down-regulation of epidermal PKC activity was observed at one (6 h) of the two (6 and 48 h) experiment times. This magnetic field-associated effect observed at the 6 h end-point disappeared when data collected after 2 weeks TPA promotion were excluded; consequently, the observed effect is not deemed to be biologically significant.

It should be noted that the assays of epidermal ODC and PKC activities are subject to inherent variability that reduces the sensitivity of these biomarkers to detect co-promotional effects. For these enzyme assays it was also necessary to utilize extracts of epidermal cells pooled from all skin specimens from mice in a single treatment/sacrifice group. Such pooling of specimens precluded an evaluation of the contribution of inter-animal variation to the overall variability of the methodologies employed. The evaluation of effects on epidermal hyperplasia, monitored as increases in epidermal thickness and labeling index, did allow the evaluation of inter-animal variation; therefore, those biomarkers offer greater sensitivity to detect a co-promotional effect.

The primary experiment also included groups of mice initiated with DMBA that were exposed to the magnetic field only (Group 1) or to the ambient field only (group 5). While not the primary objective of the current study, comparison of these two groups could reveal any direct effect of the magnetic field on short-term markers of tumor promotion. Comparison of these two groups revealed that magnetic field exposure produced no statistically significant effects on the short-term biomarkers analyzed in DMBA-initiated mice. This observation is consistent with the lack of direct tumor promoting activity of magnetic field exposure on DMBA-initiated mice demonstrated by McLean et al. (18) and Rannug et al. (19) and in our recent report (6).

In summary, the experimentation reported herein provides further support for the lack of a promotional or co-promotional effect of an ELF magnetic field (2 mT, 60 Hz) on TPA-induced tumor promotion in the skin of SENCAR mice, at least under the exposure conditions employed and within the sensitivities of the early biomarkers of tumor promotion to detect effects. The lack of an effect of the 2 mT magnetic field on these early biomarkers is consistent with a similar lack of effect on tumor development reported earlier (6).


    Acknowledgments
 
Ms Sue Goetzman provided able assistance in manuscript preparation. This research was supported under EPRI contracts WO9102 and WO2965 and NIEHS Center grant ES07784.


    Notes
 
4 To whom correspondence should be addressed Email: sa83107{at}odin.mdacc.tmc.edu Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received June 17, 1998; revised October 30, 1998; accepted December 1, 1998.