* National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709; Hoffmann-La Roche, Inc., Nutley, New Jersey 07110;
Battelle Memorial Institute, Columbus, Ohio 43201;
Research Triangle Institute, Research Triangle Park, North Carolina 27709
Received May 12, 2004; accepted July 26, 2004
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ABSTRACT |
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Key Words: TCDD; Tg.AC mice; skin neoplasms.
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INTRODUCTION |
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The Tg.AC transgenic mouse is a genetically initiated, tumor promotersensitive epidermal tumorigenesis model that is being evaluated as an alternative to the traditional bioassay (Tennant et al., 1995, 1996
). The Tg.AC mouse carries a fusion gene consisting of a zeta-globin promoter, v-Ha-ras gene, and an SV-40 polyadenylation sequence (Leder et al., 1990
). In the mouse epidermal model, activation of the Ha-ras oncogene is associated with transformation of cells to an initiated state (Balmain et al., 1984
; Balmain and Pragnell, 1983
; Brown et al., 1986
). In the Tg.AC mouse, activation of the v-Ha-ras transgene has been associated with the development of epidermal papillomas in response to dermally applied tumor promoters or complete carcinogens. Treatment-related papilloma formation in Tg.AC mice occurs after a shorter duration of exposure than in normal rodents in the traditional bioassay.
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a persistent and ubiquitous environmental contaminant. Chronic exposure to TCDD induces tumors at multiple sites in both sexes of rodents (Della Porta et al., 1987; Kociba et al., 1978
; NTP, 1982a, 1982b, 2004
; Rao et al., 1988
; Toth et al., 1979
; Van Miller et al., 1977
). TCDD is a potent hepatocarcinogen in female rats (Kociba et al., 1978
; Lucier et al., 1991
; NTP, 1982
) and a potent tumor promoter in rodent skin, liver, and lung (Beebe et al., 1995
; Pitot et al., 1980
; Poland et al., 1982
). In genotoxicity assays, TCDD fails to exhibit mutational activity (Kociba, 1984
; Poland and Glover, 1979
; Turteltaub et al., 1990
; Wassom et al., 1977
). As with other non-genotoxic carcinogens, topical exposure to 166 ng TCDD/kg induces the formation of papillomas in male and female Tg.AC mice (Eastin et al., 1998
).
Chronic human exposure to TCDD occurs primarily via the oral route. Therefore, the objectives of the present study were to examine the doseresponse relationship for TCDD-induced papillomas by dermal exposure and to investigate the induction of papillomas by TCDD after oral administration. TCDD was topically applied three times a week at doses of 0, 5, 17, 36, 76, 121, 166, 355, or 760 ng TCDD/kg body weight (equivalent to average daily doses of 0, 2.1, 7.3, 15, 33, 52, 71, 152, and 326 ng TCDD/kg/day) or by gavage five times a week at concentrations of 0, 105, 450, or 1250 ng TCDD/kg body weight (equivalent to average daily doses of or 0, 75, 321, and 893 ng TCDD/kg/day) in female hemizygous Tg.AC mice for 26 weeks. The present study compares the tumor responses for two routes of exposure over a wide dose range for a compound that has been shown to be positive in the Tg.AC skin model.
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MATERIALS AND METHODS |
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Animals. Female hemizygous Tg.AC transgenic mice (2027 days of age) were obtained from Taconic Laboratory Animals and Services (Germantown, NY). Mice were housed individually and received irradiated NTP-2000 feed (Ziegler Bros., Gardners, PA) and water ad libitum under conditions of controlled temperature (72° ± 3°F), humidity (50 ± 15%), and light (12 h light /12 h dark). The mice were genotyped for confirmation of the responsive v-Ha-ras transgene by the methods of Kantz et al. (1999). Non-responder mice were not used in this study. Random distribution of mice was performed by a body weight partitioning algorithm using a Xybion Pathology/Toxicology System. Mice were distributed into nine treatment groups (n = 20) for the dermal study and four treatment groups (n = 20) for the gavage study. Mice were acclimated for 12 days prior to the start of the studies. For the dermal study, TCDD was applied to each mouse at doses of 0 (acetone vehicle), 5, 17, 36, 76, 121, 166, 355, or 760 ng TCDD/kg body weight, three times per week for 26 weeks, over a shaved standard application site, which extended from the mid-back to the interscapular area. These doses are equivalent to average daily doses of 0, 2.1, 7.3, 15, 33, 52, 71, 152, and 326 ng TCDD/kg/day. For the gavage study, mice received 0 (corn oil vehicle), 105, 450, or 1250 ng TCDD/kg body weight, 5 days a week, for 26 weeks. These doses were equivalent to average daily doses of 0, 75, 321, and 893 ng TCDD/kg/day. Clinical observations and body weights were recorded weekly. The observation of a papilloma was not confirmed for a given mouse until the mass was observed for three consecutive weeks, at which time the mass was identified as a papilloma. Any new masses that appeared during or after this three-week confirmation period for a given mouse were also recorded as papillomas. Mice were euthanized 24 h after the last dosing by asphyxiation with CO2. A complete gross necropsy was performed on all mice. Tissues were removed, weighed, sectioned, and fixed in 10% neutral buffered formalin and examined microscopically. Samples of liver and lung tissue from TCDD-treated and vehicle-treated mice from the dermal and gavage studies were frozen in liquid nitrogen and stored at 70°C.
Histopathology. Tissues were fixed in 10% neutral buffered formalin and paraffin embedded. Embedded tissues were sectioned at 5 microns, mounted on glass slides, and stained with hematoxylin and eosin (H & E). Representative sections of tissues were examined microscopically. Non-neoplastic lesions were graded when appropriate using a semi-quantitative scale of 04, where 0 = within normal limits; 1 = minimal alteration, barely exceeding normal variation; 2 = mild, easily seen but of negligible biologic impact; 3 = moderate, of large size or potential biologic impact; and 4 = marked, essentially maximal severity.
TCDD tissue analysis. Liver, abdominal fat, and skin from application and non-application sites were collected approximately 24 h after the last dose from all control and TCDD-treated mice that survived to the terminal necropsy for TCDD tissue analysis. No TCDD concentration analysis was performed on tissues from mice not surviving to terminal necropsy. At necropsy, liver, abdominal fat, application site skin, and inguinal skin were stored at 70°C until shipped for analysis to Research Triangle Institute (Research Triangle Park, NC).
Liver samples were initially homogenized with saturated aqueous sodium sulfate. All tissue samples were spiked with an internal standard solution of 13C12TCDD (Cambridge Isotopes, Andover, MA). Skin and adipose samples were digested with aqueous potassium hydroxide solution (50%) for 15 min in a water bath (40°C) and then mixed overnight on a horizontal shaker. Samples were extracted three times with hexane/acetone (20%/80% for adipose and skin and 10%/90% for liver). Extracts were combined, reduced to dryness under nitrogen in a water bath (
40°C), and reconstituted with hexane. The adipose extracts were filtered through Whatman 0.45-µm nylon syringless filters onto pre-rinsed open bed silica gel columns (60200 mesh). Skin and liver extracts were transferred to an open bed column containing activated silica gel and anhydrous sodium sulfate. TCDD was eluted from the column with hexane, collected in a Kuderna Danish concentrator tube, and then concentrated in a water bath (
40°C) under nitrogen to 100 µl. The concentrate was spiked with 25 µl of recovery standard of 1,2,3,4,6,7,8,9-octachlorodibenzo-p-dioxin (OCDD) (Accustandard Inc., New Haven, CT) and brought to a final volume of approximately 200 µl with hexane. All were analyzed by low resolution GC/MS (Hewlett Packard 6890GC/Hewlett Packard 5973 MSD) using a J & W DB-5MS capillary column (30 m x 0.25 mm internal diameter; 0.25-µm film) with positive electron ionization and detection in the selected ion mode (SIM).
Statistics. Organ weight data were tested for homogeneity of variance by Bartlett's test. For the data that were not homogeneous, a separate variance t-test was performed. Significant differences for organ weight data with homogeneous variance were determined by one-way analysis of variance (ANOVA) and pairwise comparisons by Dunnett's test. Dunnett's test was upper one-sided to evaluate the effect of TCDD exposure on increase compared to the control group. The Poly-k (k = 3) test was used to assess the survival-adjusted incidence of non-neoplastic lesions (Bailer and Portier, 1988; Piegorsch and Bailer, 1997
; Portier and Bailer, 1989
). Dose-related effects on papilloma incidence were analyzed by Tarone's life table trend test (Tarone, 1975
), followed by pairwise comparisons of dosed groups with the control group according to Cox's method (Cox, 1972
) for testing the equality of two groups. These statistical methods take each animal's survival time into account when comparing papilloma development (time-to-tumor and proportion of papilloma-bearing animals) across dose groups. In these analyses, the event of interest was development of at least one papilloma. Animals that died early contributed information about the presence or absence of a papilloma up until the time that they died; after that time, they were censored.
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RESULTS |
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The maximum number of papillomas was observed at 26 weeks, except in the 33 and 326 ng/kg/day groups, in which the maximum number of tumors was observed at weeks 25 and 24, respectively. The mean number of cutaneous papillomas per mouse in TCDD-treated mice with tumors ranged from 1.0 for the 2.1 ng TCDD/kg dose group to 10.9 for the 326 ng TCDD/kg dose group. The mean number of skin papillomas per mouse generally increased with increasing dose. However, the mean number of skin papillomas per mouse in the 71 ng TCDD/kg dose group was slightly lower than expected relative to the other TCDD-treated groups.
Organ weights. After 26 weeks of treatment, absolute and relative liver weights were significantly higher in TCDD-treated mice at doses of 71 ng TCDD/kg and above compared to controls (Table 1). Absolute and relative liver weight tended to be higher with increasing doses. The mean absolute liver weights for the three highest dose groups increased 14%, 20%, and 51% relative to the control group, respectively. The relative kidney weights for mice receiving 152 ng TCDD/kg and absolute and relative kidney weight for mice receiving 326 ng TCDD/kg were significantly higher than control (Table 1). Absolute spleen weights for mice receiving 52 ng TCDD/kg and absolute and relative spleen weights for mice receiving 71 or 326 ng TCDD/kg were significantly higher than control (Table 1). Absolute spleen weights were 69%, 100%, 38%, and 108% higher than control in the 52, 71, 152, and 326 ng TCDD/kg groups, respectively. Absolute and relative thymus and lung weights were similar to their respective control groups (data not shown).
Histopathology. TCDD-related histopathological findings were confined to the skin and liver. Microscopic cutaneous squamous cell papillomas were observed in all groups of TCDD-treated mice, but not in controls (Table 3). Similar to gross observations, statistically significant increases in the microscopic incidence of cutaneous papillomas were observed in mice administered doses of 15 ng TCDD/kg and above compared to control. Morphologically, the papillomas were either pedunculated or sessile, with thickened surface epidermis supported by a dense connective tissue stroma. Tumor cells were well differentiated, but focal areas of the basal cell layer showing lack of usual cell polarity were frequently seen. Surface epithelium and the epithelium surrounding hair follicles was significantly hyperplastic. The surface was usually hyperkeratotic, and the dermis usually contained varying numbers of mononuclear and polymorphonuclear inflammatory cells.
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At necropsy, forestomach papillomas were observed in control and TCDD-treated mice, with no significant differences between treated mice and controls. The incidence of forestomach tumors was 1/20, 5/20, 3/20, 2/20, 1/20, 1/20, 1/20, 3/20, and 2/20 in the 0, 2.1, 7.3, 15, 33, 52, 71, 152, and 326 ng TCDD/kg groups, respectively.
Non-neoplastic lesions of the skin at the site of application, including epidermal hyperplasia, chronic-active inflammation, and hyperkeratosis were observed in some of the TCDD-treated groups (Table 3). These changes were not significantly greater than those observed in controls.
In the liver, incidences of hepatic hematopoietic cell proliferation, hepatocyte cytoplasmic vacuolization, and centrilobular hepatocyte hypertrophy were higher in mice exposed to dermal TCDD than in controls (Table 3). Hematopoietic cell proliferation was significantly increased at doses of 52 ng TCDD/kg and above compared to controls. Hepatocyte cytoplasmic vacuolization was significantly increased in mice receiving 152 or 326 ng TCDD/kg compared to controls. Hepatocyte centrilobular hypertrophy was significantly increased at doses of 71 ng TCDD/kg and above compared to controls. The incidence and/or average severity for these lesions tended to increase as the dose level increased.
Gavage Study
Survival and body weights. Survival among TCDD-treated mice was similar to controls (Table 4). Mean body weights of all groups of TCDD-treated mice remained within 10% of control mice during the study. Mean body weights of TCDD-treated mice at the end of the study were not significantly different from controls.
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Histopathology. TCDD-related histopathological findings were confined to the skin and liver. A statistically significant increased incidence in microscopic cutaneous squamous cell papillomas and carcinomas, compared to controls, was observed in the 893 ng TCDD/kg mice (Table 6). The morphology of the papillomas was similar to that observed in mice from the dermal study. The lesions coded as carcinomas appeared to arise from within papillomas. The tumor cells had undergone malignant transformation and displayed evidence of invasion into the deeper dermis and subcutaneous tissue. The squamous cells composing the rete pegs and hair follicles tended to proliferate and enlarge downward into the dermis and then broke up and degenerated. The neoplastic squamous cells often were anaplastic and spindle-shaped, giving the appearance of stromal fibrosis or a sarcomatous proliferation. This poorly differentiated form of squamous cell carcinoma has been described in the literature (Asano et al., 1998; Mahler et al., 1998
; Weiss and Frese, 1974
). Well-differentiated cutaneous squamous cell carcinomas also were seen in several mice. Carcinomas observed from the mice in the gavage study were infrequently ulcerated, but the dermis contained varying degrees of chronic-active inflammation. Mitotic figures in these neoplasms varied from common to infrequent. Although they were locally invasive, there was no evidence of lymphoid or distant organ metastases involved with the carcinomas observed in mice from the gavage study.
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There was a single incidence of epidermal hyperplasia observed in mice in the high-dose group (893 ng/mg) (Table 6).
The incidence of hepatocellular cytoplasmic vacuolization and hepatocellular centrilobular hypertrophy in the liver was significantly higher in TCDD-treated mice receiving 321 or 893 ng TCDD/kg compared to controls (Table 6). Hematopoietic cell proliferation in the liver was also significantly increased in TCDD-treated mice at all doses compared to controls. Hepatic hematopoietic cell proliferation consisted of random, focal aggregations of hematopoietic cells throughout the hepatic parenchyma. The incidence and average severity showed a dose-related increase in TCDD-treated mice. Centrilobular hepatocellular hypertrophy was only observed in the two highest-dose groups, with the average severity being greatest in the 893 ng TCDD/kg group. Hepatocellular cytoplasmic vacuolization also showed a dose-related incidence increase. The hypertrophied hepatocytes around the central veins were the most severely affected.
Dosimetry
In the dermal study, concentrations of TCDD were determined in liver, adipose, inguinal skin, and skin at the site of application from mice at the end of the 26-week study. The lowest dose at which measurable concentrations of TCDD were observed in the liver, adipose tissue, and inguinal skin (away from the site of application) was 15 ng TCDD/kg (Table 7). In the skin at the site of dermal application, concentrations of TCDD were detectable at doses as low as 2.1 ng TCDD/kg. In all tissues analyzed, concentrations of TCDD were higher with respect to increasing doses, with the greatest tissue concentrations being observed in the 326 ng TCDD/kg group. There was a linear relationship between administered dose of TCDD and mean internal dose in the skin at the site of application (Fig. 1A).
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The number of mice with cutaneous papillomas was dose-dependently higher in TCDD-treated mice by both routes (Fig. 2). With respect to administered dose, higher doses of TCDD by gavage were required to elicit a similar incidence of cutaneous papilloma formation than in dermally treated mice (Fig. 2A). On a mean tissue concentration basis, the cutaneous papilloma incidence was higher in dermally exposed mice than in mice exposed to TCDD by gavage (Fig. 2B). The slope of the doseresponse curve for the incidence of cutaneous papillomas with respect to mean skin concentrations of TCDD was steeper in mice receiving TCDD by dermal exposure than by gavage.
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DISCUSSION |
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Dermal administration of average daily doses of 2.1, 7.3, and 15 ng TCDD/kg resulted in 5%, 15%, and 30% incidences of cutaneous papillomas, respectively; whereas oral average daily doses of 75, 321, and 893 ng TCDD/kg were required to elicit a similar response. Differences in response between oral and dermal exposure in Tg.AC mice are consistent with previous studies on the effect of oral and dermal exposure to benzene on papilloma incidence and granulocytic leukemia (French and Saulnier, 2000; Tennant et al., 1999
). Slower absorption and altered metabolism of benzene may be responsible for the route of administration differences in benzene-induced granulocytic leukemia (Hoffmann et al., 2001
). Because papilloma formation occurs at the site of application and there is minimal metabolism of TCDD in vivo, these differences are likely not a factor in the present study.
Direct exposure to the skin (the site of papilloma formation) in dermally exposed mice resulted in higher mean TCDD concentrations at the site of application compared to skin concentrations in the gavage study at the end of the 26-week studies. At similar mean terminal skin concentrations of TCDD, the incidence of cutaneous papillomas was lower in the gavage study than in the dermal study. Therefore, differences in cutaneous papilloma response between dermal and oral TCDD exposure do not reflect higher concentrations of TCDD in the skin at the end of the 26-week study.
Although data on absorption and distribution of TCDD in Tg.AC mice are not available, dermal absorption in male Fischer 344 rats is less than 50% of the administered dose (Banks and Birnbaum, 1991; Brewster et al., 1989
). Greater than 50% of the unabsorbed administered dose remains at the site of application within the lipid-rich stratum corneum, the outermost layer of the epidermis (Banks and Birnbaum, 1991
). In Tg.AC mouse skin, papillomas arise from the clonal expansion of cells around the upper end of the hair follicle in the epidermis and, possibly, a subpopulation of interfollicular cells of the epidermis (Hansen and Tennant, 1994a
, 1994b
). These studies in Tg.AC mice suggest that certain cells in the epidermis are target cells in which activation of the transgene gives rise to papilloma formation. Presumably, chemicals capable of mediating papilloma formation must reach these target cells to induce transgene expression. If the absorption kinetics of TCDD in Tg.AC mice are similar to male Fischer rats (Banks and Birnbaum, 1991
), unabsorbed TCDD on the surface of the skin and in the anucleated cells of the stratum corneum may not represent an effective dose of TCDD to target cells in the skin and likely does not contribute to the burden of TCDD in the skin capable of inducing transgene expression and papilloma formation. Skin TCDD concentrations presented in this study reflect a total amount of TCDD on the surface and in all layers of the skin, including the stratum corneum. Therefore, skin TCDD concentrations for the dermal study may include "excess" TCDD that is not reaching target cells and that likely represents an over-estimate of the skin burden available to drive papilloma formation.
There was a linear relationship between administered dose and mean concentration of TCDD in the skin at the end of the study regardless of route of exposure. However, at similar terminal skin TCDD concentrations, the incidence of cutaneous papillomas was greater in mice receiving TCDD by dermal exposure than by gavage. These data suggest that terminal skin TCDD concentrations alone may not be appropriate to describe differences in response between the routes of exposure. Instead, these differences may reflect pharmacokinetic differences over the duration of the study in the delivery of TCDD to the skin. For the development of neoplastic lesions, a sufficient tissue concentration over a sufficient period of time is required to elicit a biological response. In skin, steady-state tissue concentrations are achieved more rapidly after direct application than distribution to the skin after oral administration. For a simple one-compartment model independent of the number or interval of doses, a period of exposure equal to 3.32 chemical half-lives is required to achieve 90% steady-state concentrations; and a period of 6.62 half-lives is required to achieve 99% steady-state concentrations (Perrier and Gibaldi, 1982). Although the half-life of TCDD has not been determined in the Tg.AC mouse or the FVB parental strain, half-lives in other strains of mice range from 11 to 24 days (Gasiewicz et al., 1983
). Using this calculation to determine steady-state in the skin, oral exposure to TCDD for a duration ranging from 5 to 11 weeks is required to achieve 90% steady-state, and 10 to 23 weeks is required to achieve 99% steady-state, dependent upon the half-life of TCDD in the Tg.AC mouse. Because sustained skin concentrations of TCDD are likely required to induce papillomas, it would be expected that at similar doses of TCDD, papillomas would arise sooner and with a greater incidence in mice administered TCDD dermally than in those exposed by gavage. In the current studies, the time to first appearance of papillomas was 11 weeks sooner in mice dermally administered 71 ng/kg/day than in those orally administered 75 ng/kg/day. Similarly, the time to first appearance of papillomas was 6 weeks earlier in mice dermally administered 326 ng/kg/day than in those orally administered 321 ng/kg/day. These data suggest that induction of papillomas is a function of exposure concentration and duration of exposure in the skin.
A dose-dependent increase in the incidence of cutaneous squamous cell carcinomas was observed after exposure to both dermal and oral TCDD. Differences in cutaneous squamous cell carcinoma development between oral exposure and dermal exposure were not as pronounced as differences in cutaneous papilloma formation. The incidence of cutaneous papillomas was generally higher than the incidence of cutaneous squamous cell carcinomas, as is typically the case with the Tg.AC mouse. In mouse skin, papillomas may progress into invasive squamous cell carcinomas (Rehm et al., 1989). This malignant conversion of papillomas to carcinomas spontaneously occurs at a low rate, but it can be significantly increased by exposure to genotoxic or tumor-promoting agents (Hennings et al., 1993
; Owens et al., 1995
; Yuspa, 1994
). The relationship between the incidences of cutaneous papillomas and squamous cell carcinomas in the dermal study (Fig. 4) demonstrates that increased cutaneous papilloma incidence correlates with an increase in cutaneous squamous cell carcinoma incidence. These data suggest that exposure to dermal TCDD induces the progression of benign papillomas to malignant squamous cell carcinomas. However, the incidence of squamous cell carcinomas at 893 ng TCDD/kg/day in the gavage study was 13/20 compared to 11/20 for histopathologically observed cutaneous papillomas. These data suggest that either oral exposure to TCDD drives cutaneous squamous cell carcinomas development at a higher rate than by dermal exposure or that the pathogenesis of these skin lesions may not represent a continuum between papillomas and carcinomas.
These studies demonstrate that neoplastic changes and the induction of skin cancer in the Tg.AC mouse are a response to systemic exposure and not solely a local response at the site of dermal application. They also suggest that the Tg.AC mouse model is more responsive to TCDD-induced cutaneous papillomas and squamous cell carcinomas of the skin by dermal exposure than by oral exposure. These differences may be a result of different exposure levels/rate of the target cells in the skin where transgene activation occurs. A clearer understanding of pharmacokinetic and pharmacodynamic differences between oral and dermal exposure requires further investigation. The data from these studies provide the opportunity to compare the relative sensitivity of the tumor response in the Tg.AC mouse with the response in the traditional 2-year rodent bioassay, and to determine whether extrapolated human risks are similar using tumor formation for doseresponse modeling.
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ACKNOWLEDGMENTS |
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NOTES |
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1 To whom correspondence should be addressed at National Institute of Environmental Health Sciences, MD D4-01, P.O. Box 12233, Research Triangle Park, NC 27709. Fax: (919) 541 4704. E-mail: walker3{at}niehs.nih.gov.
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