Susceptibility of proliferating cells to benzo[a]pyrene-induced homologous recombination in mice
A.J.R. Bishop,
B. Kosaras1,,
N. Carls,
R.L. Sidman1, and
R.H. Schiestl2,
Department of Cancer Cell Biology, Harvard School of Public Health, 665 Huntington Avenue, Boston, MA 02115, USA and
1 Department of Neurosurgery, Brigham and Women's Hospital, LMRC 221 Longwood Avenue, Boston, MA 02115, USA
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Abstract
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The pink-eyed unstable mutation, pun, is the result of a 70 kb tandem duplication within the murine pink-eyed, p, gene. Deletion of one copy of the duplicated region by homologous deletion/recombination occurs spontaneously in embryos and results in pigmented spots in the fur and eye. Such deletion events are inducible by a variety of DNA damaging agents, as we have observed previously with both fur- and eye-spot assays. Here we describe a study of the effect of exposure to benzo[a]pyrene (B[a]P) at different times of development on reversion induction in the eye. Previously we, among others, have reported that the retinal pigment epithelium (RPE) displays a position effect variegation phenotype in the pattern of pink-eyed unstable reversions. Following an acute exposure to B[a]P or X-rays on the tenth day of gestation an increased frequency of reversion events was detected in a distinct region of the adult RPE. Examining exposure at different times of eye development reveals that both B[a]P and X-rays result in an increased frequency of reversion events, though the increase was only significant following B[a]P exposure, similar to our previous report limited to exposure on the tenth day of gestation. Examination of B[a]P-exposed RPE in the present study revealed distinct regions where the induced events lie and that the positions of these regions are found at increasing distances from the optic nerve the later the time of exposure. This position effect directly reflects the previously observed developmental pattern of the RPE, namely that cells in the regions most distal from the optic nerve are proliferating most vigorously. The numbers and positions of RPE cells displaying the transformed (pigmented) phenotype strongly advocate the proposal that dividing cells are at highest risk to deletions induced by carcinogens.
Abbreviations: B[a]P, benzo[a]pyrene; dpc, days post-coitum; dpp, days post-partum; RPE, retinal pigment epithelium.
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Introduction
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Over the last decade, an increasing number of reports have suggested that gross genomic deletions are the cause of various diseases. Molecular analysis has revealed that such deletion events were often mediated by regions of shared homology (111). Perhaps the most prevalent example of these events is in carcinogenesis, when an initial mutation in a tumor-suppressor gene is revealed by loss of heterozygosity, often occurring by deletion of the functional homolog (12). In the last decade our laboratory has examined homologous deletions in a number of different systems, such as the yeast DEL assay (1316) or the mammalian DEL assay (17). These systems are highly inducible by a wide variety of carcinogens, a finding similar to early work by Becker (1820) who demonstrated that recombination was inducible in Drosophila. More recently, other laboratories have demonstrated that systems more similar to those developed in our laboratory display the same phenomenon (2123). The current study is designed to further our understanding of this process.
The murine pigmentation gene, pink-eyed, p, encodes a melanosomal integral membrane protein (2427) that controls the biogenesis of melanosomes (28). In the absence of a functional p gene, mice have pink eyes and dilute coat color (29). The recessive mutation used in this study, pink-eyed unstable, pun, is the result of an internal tandem duplication of 70 kb that disrupts the wild-type p transcript with extra exons (30,31). The pun mutation spontaneously reverts to wild-type by deletion of one copy of the 70 kb repeat (30,31). When a deletion/reversion event of pun occurs somatically in a precursor of a melanocyte or retinal pigment epithelium (RPE) cell, the reverted cell may proliferate and differentiate into a clone of pigmented cells. Such patches or spots have been observed in both the fur (30,32) and eyes (3335) of pun mice. Molecular analysis has confirmed that these events are the result of pun reversion (3032). On the C57BL/6J inbred background, ~510% of pun mice spontaneously display visible fur spots (30,32) and four to five eye spots are observed per RPE (35,36).
The RPE of pun mice is composed mainly of transparent (unpigmented) cells and a few well-pigmented ones (eye spots). Previously, we demonstrated that acute exposure to benzo[a]pyrene (B[a]P) or X-rays on the tenth day of development increased the frequency of reversion events in a distinct region of the adult RPE (36). Here we examine exposure at several stages of eye development and reveal that both B[a]P and X-rays result in an increased frequency of reversion events, though only B[a]P significantly so, similar to our previous report (36). Further analysis of B[a]P-exposed RPE revealed that induced events were found in distinct regions varying systematically according to the developmental stage of exposure. The position of this region of induction was at an increasing distance from the optic nerve the later the time of exposure. These results reflect the developmental pattern of the RPE and indicate a particular susceptibility of proliferating cells to DNA damage-induced homologous recombination.
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Materials and methods
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Mice
C57BL/6J-pun/pun mice were obtained from The Jackson Laboratory (Bar Harbor, ME). Mutant homozygotes are fertile, were bred inter se in the institutional animal facility under standard conditions with a 12 h light/dark cycle, and were fed a standard diet and water ad libitum. Pregnancy was timed by checking for vaginal plugs, with noon of the day of discovery counted as 0.5 days post coitum (dpc). Similarly, the time of birth of a litter was timed with the noon of discovery counted as 0.5 days post partum (dpp).
Exposure to agents
Animals were either injected interperitonally with 150 mg/kg of B[a]P (Sigma) (CAS no. 50-32-8) or irradiated with 1 Gy of X-rays. Alternatively, newborn pups were fed the equivalent dose of B[a]P. B[a]P was dissolved in corn oil at a concentration of 22.5 mg/ml such that 0.2 ml was used per 30 g mouse. Irradiation was performed in a Philips MCN 165 industrial X-ray generator operated at 160 kVp and 18 mA at a dose rate of 0.74 Gy/min. Mice were exposed in individual sterile polypropylene containers. The dose of radiation delivered was checked using a Victoreen C-r 570 meter.
Dissection of the RPE
Eyes taken from C57BL/6J pun /pun mice killed at 20 days-old were processed to expose the RPE layer as previously described (36,37). The eye was removed from its orbit and immersed in fixative (4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4) for 1 h and then in phosphate-buffered saline until dissection. An incision was made at the upper corneo-scleral border to allow removal of the cornea and lens. To flatten the eye-cup, six to eight incisions were made from the corneo-scleral margin towards the centrally positioned optic nerve, and the dissected eye-cup was placed on a glass slide with retina facing up. The retina was then gently removed and the residual specimen consisting of sclera, choroid and RPE, with RPE facing up, was mounted in 90% glycerol for microscopic analysis.
Scoring a single reversion event, visualized as an eye-spot
We defined two or more adjacent pigmented cells, or pigmented cells separated from each other by no more than one unpigmented cell, as an eye-spot that resulted from one reversion event (37). The number of eye spots in each RPE and the number of cells that comprised each eye spot were counted. Positions of eye spots were mapped.
Microscope, digital camera and software
The RPE was scanned with a DC120 digital camera (Eastman Kodak Company) mounted on a DMLB microscope (Leica Microsystems) using a 2.5x N-plan objective. The images were assembled and examined in Adobe Photoshop 5.0 on a Macintosh Power Computer. All data were stored and processed with Microsoft Excel 98.
Distance analysis of eye spots from the optic nerve
Spots were identified under the microscope and compared with their scanned digital images. Distances were measured with the Adobe Photoshop 5.0 Measurement Tool. Distances were converted from pixels to millimeters by counting the number of pixels/mm on the image of a micron scale reticule scanned at the same optical settings as the RPE. Two distances were measured for each eye spot: the `eye-spot distance' is the distance from the center of the optic nerve head to the most proximal edge of the eye spot, and the `RPE distance' of an eye spot is the distance from the optic nerve through the eye spot to the outer edge of the RPE. Dividing the eye-spot distance by the RPE distance gives the proportional distance of each eye spot from the outer edge of the RPE, or its position. The position of each eye spot was determined in this manner to compensate for minor differences in the size of the eyes.
Statistical analysis
Comparison between numbers of events was done by a standard G test (38). The G test is equivalent to a contingency
2 test but allows for classes with zero events.
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Results
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Induction of eye spots
Previously, we demonstrated that pun reversion events are induced in the RPE when pregnant mice are exposed at 10.5 dpc to B[a]P or X-rays (36), similar to previous work with the fur assay with these animals (32,39). Here we examined pun reversion in the RPE following exposure at different times of development to either B[a]P or X-rays. The RPE of the resultant litters were examined for the number of eye spots present (Figure 1
). As previously reported for both the fur- and eye-spot assay B[a]P is a potent inducer of deletions (32) whereas X-rays are less potent (36,39). For each of the different times, B[a]P increased the number of eye spots significantly in comparison with the spontaneous frequency. Similar to our previous results, exposure to X-rays resulted in an increase in the number of eye spots following each of the different times of exposure, but that increase was not significant (Figure 1
).

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Fig. 1. Average number of eye spots found per RPE examined following either B[a]P or X-ray exposure at different dpc or dpp. Unexposed control frequency is also shown, with the error bars representing one standard deviation. The solid line across represents the mean of the control and the broken line one standard deviation of the control.
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Number of reverted cells and size of eye spots
To gain further insight into the nature of the induction of pun reversion in the RPE we selected RPEs from 8.5, 12.5 and 17.5 dpc B[a]P exposures for further analysis. In the previous report, we examined the number of pigmented cells that comprise each eye spot for 32 unexposed and 14 B[a]P 10.5 dpc exposed RPEs (36); these data have been incorporated into the present study for comparison. The total number of reverted cells in each RPE were determined (Table I
). The most interesting result is a highly increased number of reverted cells following exposure to B[a]P at 8.5 dpc, which is reflected in the increased average spot size in these RPE. Figure 2
displays the most dramatic effect where a 217 cell-sized eye spot has been produced and follows a radial pattern out from the optic nerve head. In Figure 3
the frequency of each size eye spot (e.g. one pigmented cell, two pigmented cells, etc.) per RPE is displayed. Irrespective of the developmental stage of exposure to B[a]P, the frequency of each size eye spot is increased compared with the spontaneous frequency (Figure 3
). But more significantly, as shown in Table II
, the frequency of large eye spots (greater than three cells) is significantly increased in the 8.5 dpc exposed RPE compared with the control as well as the later exposures.

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Fig. 2. (A) A 8.5 dpc B[a]P exposed RPE. Clearly visible is a large eye spot and a multitude of smaller eye spots extending radially from the optic nerve to the edge of the RPE. The largest eye spot consists of 217 reverted cells, which is magnified in panel (B).
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Fig. 3. The frequency of different size eye spots that arise spontaneously or following particular times of exposure to B[a]P.
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Distance effect of induction
The position of each eye spot was determined in each adult RPE after B[a]P exposure at 8.5, 10.5, 12.5 and 17.5 dpc. Each `position' is a relative measurement, where `1' is a concentric zone at the edge of the RPE, ~2.6 mm from the optic nerve, and `0.1' is the concentric zone closest to the optic nerve head (36). We found an increased frequency of eye spots in all 10 concentric zones after all four times of exposure to B[a]P, with the exception of 17.5 dpc exposure for the 0.1 zone, closest to the optic nerve head (Figure 4
).

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Fig. 4. The frequency of eye spots in different regions of the RPE that arise spontaneously or following particular times of exposure to B[a]P. A position of 0.0 is equivalent to the optic nerve head, whereas a position of 1.0 is at the edge of the RPE.
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Previously, we examined in detail the pattern of eye-spot distribution following B[a]P exposure at 10.5 dpc (36). In that study we reported that the majority of the spontaneous eye spots were located in the distal part of the RPE (>60% from 0.7 to 1.0), while an increased proportion of induced eye spots were located between positions 0.2 and 0.7 following the B[a]P exposure on 10.5 dpc. This zone from positions 0.20.7, with the highest signal-to-noise ratio, was termed the `region of induction' (36). Exposure to B[a]P at 8.5 dpc produced an even more dramatic region of induction in zones 0.20.6, though all regions demonstrated some level of induction (Figure 4
).
In general we found that each time of exposure resulted in two regions of induction that were immediately adjacent to each other (data not shown). Further examination revealed that these two regions generally consisted of different sized eye spots. Therefore, in Figure 5
we separately present the positions of the eye spots that were either one cell in size (`singlets') or greater than one cell in size (hereafter referred to as `larger eye-spots'). The proportion of the identified eye spots located within a position interval is shown for these RPEs. Figure 5A
clearly illustrates that singlets are differentially induced and that the region of induction was found to be more distal from the optic nerve head the later the time of exposure. We selected regions that we considered to encompass the region of induction for each time of exposure to conduct statistical analysis, the results are shown in Table III
. In summary, a significant increase in singlets was identified for exposure on 8.5, 10.5 and 17.5 dpc in regions 00.3, 0.20.4 and 0.60.9, respectively. In contrast, exposure on 12.5 dpc resulted in a region of induction between 0.4 and 0.6 that was resolvable from 8.5, 10.5 and 17.5 dpc exposures but not the spontaneous events. This suggests that a large proportion of spontaneous single cell reversion events occurs at ~12.5 dpc.

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Fig. 5. The frequency of eye spots in different regions of the RPE relative to all the other eye spots found: (A) one-cell sized eye spots only; (B) greater than one-cell sized eye spots.
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Figure 5B
and Table IV
display a similar analysis for the larger eye spots. Again, regions of induction were identified for exposure on 8.5 and 10.5 dpc in regions 0.10.4 and 0.40.7, respectively. The 12.5 and 17.5 dpc exposures did not result in a region of induction that was significantly different from the distribution pattern of the spontaneous events, although regions of induction resolvable from the earlier exposure times, 0.70.8 and 0.91.0, respectively, were identified. The overlap between the 12.5 and 17.5 dpc exposed RPE regions of induction with the location of the majority of spontaneous larger eye-spot events suggests that these spontaneous reversion events occur between 12.5 and 17.5 dpc.
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Table IV. Frequencies of eye-spots larger than 1 cell in different positions following different times ofexposure
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A final interesting result comes from the comparison of the position analyses of the one-cell and larger eye spots. The regions of induction for the larger eye spots, both induced and spontaneous, were consistently found to be more distal to the optic nerve head than the equivalent regions of induction of the singlets (compare Figure 5A
and Table III
with Figure 5B
and Table IV
). This observation suggests that there is a difference in how these two classes of event are produced.
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Discussion
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Carcinogens induce homologous deletions
Over the past decade we have demonstrated with a number of model systems that homologous deletion is induced by a wide variety of carcinogens (1315,17,32,39,40). The in vivo system that we have used to verify our in vitro model systems monitors homologous deletion events at the pun locus (32,36,39). Here we demonstrate that pun reversion is inducible in the RPE throughout its development. Further insight into the process of induced homologous deletion was gained through a detailed analysis of the B[a]P-induced events.
Dividing cells in the mouse RPE are most responsive to B[a]P
Melanogenesis begins in the RPE segment of the developing eye-cup at ~8.5 dpc (41). Induction at such an early time allows the possibility of reversion in a progenitor cell that will continue to divide throughout the development of the RPE, so that all its progeny will display the reverted (pigmented) phenotype. The most dramatic example we observed is depicted in Figure 2
, where a large radial eye spot of 217 cells is clearly visible. No spot of this size has been observed either spontaneously or following exposure to carcinogen at later times. The fact that the eye spot followed a radial pattern is consistent with the radial development of the RPE away from the optic nerve head. Many similar radial eye spots, both spontaneous and following B[a]P exposure, have been observed previously, but these have always been much smaller, usually fewer than 15 cells in size (35; data not shown). This 217-cell clone may actually be larger, if the several `trailing' singlet and two-cell spots in the same radial orientation (Figure 2
) derive from the same reverted parent cell. We have defined clones conservatively as including only those pigmented cells lying within one cell diameter of another pigmented cell (35,36), but it is highly probable that a greater separation of cells of a given clone will occur simply through displacement caused by repeated cell division itself, as shown in vivo and in a computer model of RPE development (42). Similar events probably occur in the adjacent neural retina (43). As such large clones with `trailing' pigmented cells were never seen following later times of exposure, or in other studies, we have treated them as an exception of the early exposure, and maintain that our definition of a clone is valid, especially for later exposures where clones tend to be much better separated.
According to Bodenstein and Sidman (37,42), the RPE shows an edge-biased pattern of radial growth, with an increasing concentration of mitotic cells toward the outer margin at any given stage and fewer mitotic cells near the central optic nerve head. Kong et al. (44) reported that the RPE has its highest rate of mitotic division between 11.5 and 15.5 dpc, and Stephenson and Searle, found the highest mitotic index on 13.5 dpc (45). Previously, we reported that the majority of spontaneous pun reversion events were found in the distal RPE (36), similar to the report by Deol et al. (34) who noted a majority of events in the iris, which was not present in our preparations. We suggested that the observed position effect variegation was due to the larger number of dividing cells toward the periphery of the RPE, both a result of this region having the largest number of target cells and an accumulating random chance effect. It appears that the majority of the spontaneous events detected lay in between the induced events found following 12.5 and 17.5 dpc B[a]P exposure. This corresponds to the highest level of mitotic division in the RPE, again suggesting a greater sensitivity of dividing cells to homologous deletions, even spontaneously.
The RPE develops into a near spherical eye-cup (37). With an edge-biased growth pattern it follows that the largest number of dividing cells will be present when the circumference of the RPE reaches the widest point of the sphere, the equator. Thereafter, the circumference of the RPE will be decreasing, and consequently the absolute number of dividing cells at the edge of the RPE will be decreasing. This reduction in the number of dividing cells must result from an increasing frequency of terminal divisions just before the eye-cup reaches its maximal circumference. Assuming that the frequency of reversion events is directly dependent on the number of dividing cells, with an increasing chance of reversion the later in development, then we should observe the general position effect variegation that was previously reported (34,36). But, just before the equator, there should be an increased number of terminally-dividing cells, which makes it more likely that any cell that undergoes pun reversion at this time/position will be in terminal division, thus producing a bias for single-cell eye spots in this region. The pattern observed for the position of singlets recapitulates this model. We measured the equator of the eye to be ~2 mm from the optic nerve head, or at a position of ~0.7. A slight increase in singlets, observed as a shoulder in Figure 5A
, was identified in the same location as the 12.5 dpc region of induction, 0.40.8 (including both singlet and larger eye spots).
In this study we have demonstrated that acute exposure to B[a]P results in distinct regions of induced pun reversion events within the RPE. The earlier the exposure, the closer to the optic nerve head this region was found. These distinct regions follow the pattern of RPE development, with the outermost cells having the highest mitotic activity. Again these results strongly suggest that the dividing cells are the ones that are sensitive to B[a]P-induced pun reversion.
In a yeast model system the effect of cell cycle arrest on induction of homologous deletion by different carcinogens has been previously investigated. It was found that only DNA double-strand breaks induce DEL recombination in arrested cells, other forms of DNA damage such as DNA single-strand breaks, UV lesions as well as exposure to alkylating agents need DNA replication to induce DEL recombination (46,47). We previously demonstrated that acute exposure to B[a]P induced eye spots in a specific region of the RPE. Here that study was extended to show that the location of this region was dependent upon the time of exposure to B[a]P. The pattern and timing of the peak of induction follows the developmental pattern of the RPE, suggesting that those cells that are dividing at the time of exposure are most susceptible to carcinogen-induced homologous deletion, similar to our work in yeast. One possible explanation is that DNA replication is required to convert the DNA damage into recombinagenic lesions. Alternatively, the damage may lead directly to recombination. It has recently been proposed that stalled replication forks may be removed by a recombination event (for reviews see 48,49). One cause of replication fork stalling may be DNA damage, either with a single-strand break in the template producing a double-stranded one or conversion of a stalled replication fork into a structure similar to a Holliday junction. A B[a]P adduct on one strand of the replicating DNA may result in a stalled replication fork and recombination, resulting in pun reversion that segregates into a single daughter cell. If this occurs at the periphery of the growing front, this reverted cell is more likely to replicate to form a larger eye spot. The distribution of reverted cells in distinct regions of the RPE, according to the time of administration of the carcinogen, is consistent with a dependence of B[a]P induction of homologous recombination on cell division. This model fits both our observations in yeast and those reported here.
Homologous recombination as potential mechanism of carcinogenesis
Homologous recombination may play a key role in carcinogenesis. Actively dividing cells are thought to be the most prone to developing cancer, and in this study it appears that only those cells that are actively dividing at the time of exposure are susceptible to induced homologous recombination. This observation is comparable to a recent study that elegantly demonstrated that DNA repair of mutation also appears to require DNA synthesis and is consequently higher in actively dividing cells (50). Mitogenesis has been proposed to be an important contributor to carcinogenesis (51,52) as evidenced by a higher risk for cancer after regeneration or in children. Furthermore, chemical carcinogenesis and transformation are most efficient if the target cells are treated just prior to or during S-phase (53,54). In summary, the facts that (i) differently acting carcinogens induce homologous deletions, (ii) a higher than normal frequency of homologous deletion is found in cells from patients with mutations resulting in an elevated occurrence of cancer and (iii) proliferating cells are more prone to homologous deletions and development of cancer, all emphasize the correlation between homologous DNA deletions and carcinogenesis (for a review see 55). Aberrant homologous recombination, as assayed here, can occur naturally between the many repeated DNA elements in mammalian cells and is highly likely and potentially deleterious. In fact there are a number of genetic diseases where cells from the patients display an elevated level of genomic instability and the patients have a higher than normal predisposition to cancer, for example, Ataxia telangiectasia (56), Li-Fraumeni syndrome (57), Blooms syndrome (58), Werners syndrome (59), Cockayne's syndrome, Fanconi's anaemia, Lynch syndromes I and II, WiscottAldrich syndrome and xeroderma pigmentosum (60). For one of these diseases, Ataxia telangiectasia, we have demonstrated that the mouse model does indeed display an increased spontaneous frequency of homologous recombination (61). In addition, in some cases where the genetic change causing cancer has been characterized on the molecular level, recombination events between homologous sequences have been identified. This is true for acute myeloid leukemia (10) and chronic myelogenous leukemia in the Philadelphia chromosome translocation (62,63). Thus, it seems likely that homologous recombination plays a fundamental role in the development of a cancer cell. For this reason, the pun mouse RPE appears to be a good system for measuring the potential of carcinogens to induce deletions by homologous recombination, an event that may be a key step in carcinogenesis.
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Notes
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2 To whom correspondence should be addressed Email: schiestl{at}hsph.harvard.edu 
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Acknowledgments
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We thank the R.H.S. laboratory members for their comments. This work was supported by grants from the US Environmental Protection Agency, National Center for Environmental Research and Quality Assurance No. R 825359, National Institute of Environmental Health Sciences, NIH, RO1 grant no. ES09519 and KO2 award ES00299 (to R.H.S.) and NIH RCDA award no. F32GM19147 (to A.J.R.B.).
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Received October 26, 2000;
revised December 15, 2000;
accepted December 20, 2000.