Monsanto Company, St. Louis, Missouri 63167
Received October 1, 1999; accepted January 3, 2000
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ABSTRACT |
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Key Words: alachlor ethane sulfonate; alachlor; metabolite; pharmacokinetics; common mechanism; oncogenic potential; risk assessment.
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INTRODUCTION |
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Alachlor produces nasal, thyroid, and glandular stomach tumors in rats by non-genotoxic, threshold-sensitive processes. Mechanistic investigations have identified specific pharmacokinetic and pre-neoplastic effects that are necessary for the subsequent development of tumors. Nasal tumors are produced as a result of species- and tissue-specific metabolism, nasal protein binding, and sustained cell proliferation (Heydens et al., 1999). The stomach tumors occur as a result of substantial mucosal atrophy followed by a marked proliferative response (Thake et al., 1995
). Thyroid tumors of the follicles result from changes including the induction of hepatic uridine diphosphate glucuronosyl transferase (UDPGT), increased thyroid hormone elimination, and excessive TSH stimulation. As would be expected with such a mechanism, increased liver and thyroid weights are consistently observed during alachlor administration (Wilson et al., 1996
).
The pre-neoplastic changes described above develop as early as 1460 days after dosing begins and are sustained upon prolonged administration. Therefore, liver, thyroid, nasal and stomach tissues from the 91-day ESA study in rats were examined to determine if the metabolite produces the same pre-neoplastic effects seen with alachlor. In addition to these investigations, metabolism and whole body autoradiography (WBA) data were generated in a separate study. Results from all these studies allowed an assessment of the oncogenic potential of ESA relative to alachlor and the determination of whether ESA shares a common oncogenic mechanism with alachlor.
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MATERIALS AND METHODS |
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Animals and husbandry.
Male and female Long-Evans rats were obtained from Charles River Breeding Laboratories (Portage, MI). At study initiation, the animals were approximately 6- (males) or 8 (females)-weeks-old.
Experimental design.
Four male and 4 female Long-Evans rats were given a single dose of radiolabeled ESA by oral gavage at a target dose level of 70-mg/kg body weight (bw). This dose was selected to permit direct comparison between alachlor and ESA, since it is the same dose level used in alachlor metabolism/whole body autoradiography studies. The test material was administered in a vehicle of 5% water/corn oil at a dose volume of 4-ml/kg bw. Urine, feces, and cage washes were collected from all animals. Two males and two females each were sacrificed at both 24 h and 5 days after dosing. All animals were processed by whole-body autoradiography to determine tissue distribution.
Urine, feces, and cage washes were collected from all animals at 24 h after dosing and daily thereafter until the animals were terminated. Urine and feces were kept chilled during collection. Roth-type metabolism cages were rinsed with distilled water at each collection period. At the end of the study, each cage unit was rinsed thoroughly with distilled water and then with 70% acetonitrile in distilled water. The radioactivity in the cage washes was quantitated.
The fecal collections were homogenized with water using a Tekmar tissuemizer (Tekmar Instruments, Cincinnati, OH) and weighed. Duplicate aliquots of the fecal homogenates were combusted in a Packard System 387 Sample Oxidizer (Packard Instrument Co., Downers Grove, IL). The scintillation cocktail was a mixture of Permaflour® and Carbosorb® Oxidizer (Packard Instrument Co.). Duplicate aliquots of the urine (~0.10.3 g) and cage wash samples (~0.10.5 g) were weighed and analyzed directly by liquid scintillation counting (LSC) after adding approximately 15 ml of Ultima Gold® counting cocktail (Packard Instrument Co.). The urine, feces, and cage washes were analyzed separately for each rat at each collection period.
The 24-h urine samples from each animal were individually analyzed for the presence of ESA and possible metabolites. Analyzing the 24-h fecal samples from animals sacrificed on day 1, and the pooled 048-h fecal samples from animals subsequently sacrificed at day 5, characterized radioactivity in the feces. All fecal samples were extracted 3 times with 60% aqueous acetone and concentrated under a gentle stream of nitrogen prior to analysis.
High pressure liquid chromatography (HPLC), coupled with radioactive flow monitoring (HPLC/RAD) and subsequent fraction collection, was used to profile all the urine and fecal samples. The HPLC system used for chromatographic profiling consisted of a Varian 5500 HPLC with a variable wavelength detector, a Beckman Model 171 Radioactivity monitor, and a Gilson Model 202 fraction collector. The radioactive flow monitor was connected downstream in series with the UV detector. A Beckman ODS Ultrasphere column (5 micron packing, 10 mm x 25 cm) was employed for the profiling. The solvent system utilized 2 mM (NH4)2HPO4 and methanol in the following gradient: 0100% methanol over 30 min in a linear gradient at 4 ml/min. The HPLC effluent was collected at 0.5-min intervals, mixed with Packard Ultima-FloTM M scintillation cocktail at a 1:2.5 ratio, and passed through the 2750-µl liquid cell of the rad-monitor. An authentic standard of ESA was prepared and the identity confirmed by liquid chromatography/mass spectrometry (LC/MS).
For whole-body autoradiography analysis, animals were sacrificed by CO2 inhalation and rapidly frozen in a hexane/dry ice bath at approximately 70°C. The frozen animals were embedded in carboxymethylcellulose, mounted in a Leica Cryomacrocut (Leica, Deerfield, IL), and sectioned sagittally at a thickness of 40 µm. These sections were dehydrated in the cryostat for at least 24-h and then pressed onto x-ray film. Following exposure, the films were developed in Kodak GBX developer and fixed in Kodak Rapid Fixer.
Mechanistic Evaluations: 91-Day Toxicity Study
In-life exposure.
Specimens used for the evaluations described below were obtained from a study conducted previously in male and female Fischer 344 rats (Charles River Laboratories, Inc., Raleigh, NC). In that study, groups of 10 male and 10 female rats received ESA in drinking water at target concentrations of 200, 2000, and 10,000 ppm for 91 days. These levels corresponded to doses of: 16, 157, and 896 mg/kg/day, respectively, for males; 23, 207, and 1108, respectively, for females. A concurrent control group received untreated drinking water under the same experimental conditions. Organ weights and tissue samples were obtained at the terminal necropsy. Additional details on the conduct of the study are reported elsewhere (Heydens et al., 1996a).
Evaluation of tissues.
Nasal tissues from control and 2000-ppm dose level male rats were examined. This level was selected for evaluation because the quality of most of the preserved nasal tissue specimens at the 10,000-ppm level was inadequate. The 2000-ppm level (157 mg/kg/day for male rats) exceeded the highest level tested in the alachlor bioassays (126 mg/kg/day), a dose which induced nasal tumors and has been shown to produce a sustained, significant increase in cell proliferation. Male rats were examined because alachlor-induced nasal tumors occur more frequently in males than females.
At the time of necropsy, the nasal turbinates were completely flushed with 10% neutral buffered formalin and the entire head was then immersed in fixative. The wet tissue of the nasal cavities was subsequently decalcified and trimmed. The tissue sections were processed and then embedded in paraffin. The nasal cavity tissue used in these evaluations was taken from the olfactory region at the level of the second palatal ridge (Level III). This is the area of the nose where alachlor-induced tumors are found. Paraffin blocks of the nasal tissues were sectioned at a thickness of 4 µm for subsequent determination of cell proliferation by the proliferating cell nuclear antigen (PCNA) technique. Cell proliferation was measured using PC10 antibodies (Dako) to PCNA followed by biotinylated anti-mouse IgG antibodies and streptavidin-horseradish peroxidase. The staining reaction was then detected by use of the chromogen, diaminobenzidine, and a hematoxylin counterstain. Tissues from 5 control and 9 ESA-treated animals had adequate staining quality for analysis of cell proliferation. The septum and olfactory turbinates from each animal were evaluated separately. The animals were examined without knowledge of the dose group from which they originated. A standard pattern was used to count the labeled nuclei, which included 10 sites for the septum and 16 sites for the turbinates. Observation of labeled nuclei was determined using a square eyepiece graticle with a 40x objective (total magnification of 400x). Results were expressed as the number of labeled nuclei per millimeter of mucosal length.
Stomach tissues from control and the 10,000-ppm dose level (1108 mg/kg/day) female rats were evaluated for cell proliferation and mucosal thickness. Female rats were examined because alachlor-induced stomach tumors are preferentially produced in this sex. The PCNA technique was used to evaluate cell proliferation in glandular stomach mucosal tissue. Monoclonal PC10 (BioGenex) antibodies to PCNA were used according to methods described previously (Dietrich, 1993; Galand and Degraef, 1989
). The detection system was biotin-streptavidin (BioGenex). The staining reactions were visualized with diaminobenzidine on a background of hematoxylin counterstain.
The numbers of labeled cells were determined in the fundus of the glandular stomach using a square graticule (Cambridge Instruments, Inc.) with 25 equal subdivisions at 400x magnification. Each side of the graticule was confirmed by measurement with a second micrometer (Graticules, Ltd.) as 0.125 mm, providing a square counting area that usually contained between 50 and 90 cells at this magnification. All labeled nuclei in the area were counted for PCNA. Where possible, counts were obtained for longitudinal sections of fundus from 1 to 3 separate slides for each animal. In the fundus, 2 levels were scored: the neck region and the basal mucosa. For the former, the graticule was placed with its center over the proliferative zone in the neck region. For the deeper level, the bottom side of the graticule was aligned over the basal edge of the mucosal epithelium. The graticule was then positioned at least one width along the mucosa from each preceding site until 10 graticule areas were counted at each fundic level. In this way, between 450 and 1000 fundic nuclei were counted per fundic level per slide. Only glandular epithelial cells were counted.
To evaluate fundic mucosal thickness, the full depth of gastric fundic mucosa was measured from the foveolar surface to the base of the gastric glands in longitudinal sections of tissue with the graticule described above. The limit of measurement was 0.1 of the graticule side, i.e., 0.0125 mm. Where possible, measurements were taken at 10 sites in each longitudinal section of stomach, from 1 to 3 slides for each animal.
At the conclusion of the 91-day toxicity study, the liver and thyroid (with attached parathyroids) from each animal were weighed before being placed in 10% neutral buffered formalin. The thyroids were processed and stained for subsequent histologic examination. Only data from male rats are considered here, since alachlor did not produce thyroid tumors in females.
Statistical Methods
Student's t-test was used to detect statistical differences in cell proliferation and mucosal thickness between control and treated animals. A one-way analysis of variance (ANOVA) was used to analyze liver and thyroid weights (Snedecor and Cochran, 1967).
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RESULTS |
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A representative whole-body autoradiograph is presented in Figure 2. At 24 h after dosing, the major areas of radiolabel localization were the stomach and intestinal contents. Very slight localization was present in the liver of one animal. No other sites of localization were observed.
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DISCUSSION |
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The HPLC profiles of radioactive metabolites from the feces showed very simple metabolic profiles. On average, 93% of the fecal-contained radioactivity (72% of the administered dose) was present as unmetabolized ESA. The HPLC profiles from the urine samples were also relatively simple. In males, ESA was the major peak present, representing ~41% of the urine-contained activity (UCA). Three metabolites were also present in these samples, ranging from 1026% of the UCA. The urine from females also contained ESA as the major peak present (>90% of UCA), along with very small amounts (~0.64% of UCA) of the 3 metabolites. Because a low amount of the dose was excreted in the urine, none of the ESA-derived metabolites was present at greater than 4% of the administered dose. Thus, the HPLC analyses of urine and feces demonstrated that the majority of radioactivity was excreted as parent material along with a very low level of metabolites.
The metabolic profile for ESA is in marked contrast to that observed with alachlor. In the case of alachlor, metabolism is extensive and produces a significant number of metabolites (>30) (Heydens et al., 1999). These metabolites result from initial hepatic metabolism to glutathione and glucuronide conjugates, followed by biliary excretion, gut microflora metabolism, and enterohepatic circulation. Subsequent work has shown that these processes result in the production of a diethylbenzoquinone imine (DEIQ) metabolite of alachlor. The DEIQ-protein adduct was shown to be the major alachlor-derived protein adduct binding to rat nasal turbinates, and this metabolite is believed to be responsible for the subsequent oncogenic response in that tissue (Heydens et al., 1999
).
The results of the WBA evaluation showed that almost all of the orally administered ESA and/or its metabolites remain in the gastrointestinal tract, and extremely low levels are delivered systemically. This pattern of tissue distribution is in sharp contrast to the localization observed after alachlor administration. Analysis of rats one day after alachlor administration showed localization of activity in liver, kidney, lung, stomach, intestine, and nasal turbinates (Feng et al., 1990). The localization of radioactivity in the nasal turbinates was very pronounced. Five days after dosing, localization was still present in liver, kidney, lung, intestine, and nasal turbinates. The intense localization in the nasal turbinates following alachlor administration is of special interest because of the oncogenic response observed in that tissue in the chronic bioassay. As noted above, DEIQ is the major alachlor metabolite binding to nasal tissue and is believed to be responsible for the subsequent tumor development.
In contrast to the pronounced localization and binding observed after alachlor exposure, the complete absence of any ESA-derived localization in the nasal turbinates is especially striking (Fig. 4). The absence of localization following ESA administration is consistent with the low degree of absorption observed in the metabolism portion of the study. Most importantly, this absence of binding demonstrates that target tissue exposure, and thus the potential for tumor development, is greatly reduced (or even essentially non-existent).
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ESA did not increase nasal cell proliferation in male rats treated for 91 days at 2000 ppm (157 mg/kg/day). This dose exceeded the highest level tested in the alachlor bioassays (126 mg/kg/day), a dose which produced increases in nasal cell proliferation (320379%; see Fig. 5) and tumors. Therefore, the lack of proliferation in this study supports the conclusion that ESA is unlikely to be oncogenic in the nasal tissue of rats.
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ESA produced only a minimal increase in cell proliferation in one fundic region and no decrease in mucosal thickness following administration to female rats for 91 days at 10,000 ppm (1108 mg/kg/day). This dose is approximately 5 times higher than the butachlor dose which produced dramatic changes in mucosal thickness and regenerative cell proliferation (Figs. 6a and 6b) prior to tumor formation; it is also approximately 9 times higher than the dose that produced stomach tumors in the alachlor bioassay. These results strongly support the conclusion that the potential of the ESA metabolite to produce glandular stomach tumors in rats is substantially less than that of alachlor. Based on the previous work with butachlor and alachlor, the minimal change seen in the present study indicates that even very high ESA exposure is insufficient to drive the production of stomach tumors in rats.
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Dietary administration of alachlor to male rats at 126 mg/kg/day for 28120 days produced increases in liver weights ranging from 109120% of the control values (Wilson et al., 1996). Similarly, the thyroids from animals fed alachlor for 60 and 120 days weighed 26% and 13% more than controls, respectively, and diffuse thyroid follicular cell hypertrophy and hyperplasia were readily apparent upon microscopic examination of the glands. ESA administration for 90 days at dose levels up to 896 mg/kg/day did not produce any of these effects. While these parameters are not direct measurements of the liver enzyme induction and TSH stimulation of the thyroid observed after alachlor treatment, they are sensitive endpoints which indicate the occurrence of preneoplastic changes required for subsequent tumor induction. The lack of any such changes in the liver and thyroid following subchronic, high-dose ESA administration supports the conclusion that the metabolite would not be expected to produce thyroid follicular cell tumors.
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SUMMARY AND CONCLUSIONS |
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The present work demonstrates significant differences in absorption, metabolism, excretion and tissue distribution between alachlor and its metabolite, ESA. The absorption of ESA was shown to be much lower than that of alachlor, and the metabolite was excreted more quickly than the parent material. While alachlor and its metabolites have been shown to distribute widely to various tissues systemically, ESA-derived radioactivity was confined to the stomach and intestines. It is especially important to note that alachlor administration resulted in pronounced and prolonged localization in the nasal turbinates, a tissue in which alachlor-induced tumors were observed. In contrast, there was no localization in nasal tissue following exposure to ESA. Thus, compared to alachlor, there is very limited potential for delivery of ESA to any tissues outside the gastrointestinal tract. Mechanistic investigations revealed that ESA did not cause any of the prerequisite antecedent changes which are produced by alachlor and lead to the development of the nasal, thyroid, and stomach tumors observed following chronic exposure.
Taken together, these data strongly support the conclusion that ESA is unlikely to be oncogenic in the rat. In addition, ESA does not operate via a common oncogenic mechanism with alachlor. Therefore, it would not be appropriate to include the ESA metabolite in a cumulative oncogenic-risk assessment with alachlor.
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NOTES |
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REFERENCES |
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