National Institute for Occupational Safety and Health, Morgantown, West Virginia 26505
Received April 22, 2003; accepted June 9, 2003
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ABSTRACT |
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Key Words: 3-amino-5-mercapto-1,2,4-triazole (AMT); N-(2,6-difluorophenyl)-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidine-2-sulfonamide (DE498); airway hyperreactivity; dermal sensitization; IgE.
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INTRODUCTION |
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AMT is a low-molecular-weight chemical used (1) in the synthesis of organic chemicals, (2) in the processing of silver halide photographic materials, (3) as an antioxidant for aluminum and lubricating oils, and (4) as a viscosity index improver and dispersant. To meet these usage needs, 250,000 pounds of AMT was imported into the United States in 1993 (U.S. EPA, 1998). Despite the wide potential for exposure to this chemical, there is little toxicity data available. A single study reported a decrease in thyroid peroxidase and thyroxine (T4) concentrations, with a concurrent increase in thyroid weight and thyroid stimulating hormone (TSH) levels in Fischer rats following oral exposure to AMT (Takaoka et al., 1994
). Therefore, the Environmental Protection Agency has listed AMT on the Toxic Substance Control Act (TSCA) for further evaluation of its toxicity potential (1998).
DE498 (trade name; Flumetsulam) is a broad-spectrum herbicide acting by inhibiting acetolactate synthase (ALS), an enzyme necessary for branched-chain amino acid (leucine, isoleucine, and valine) synthesis (Kleschick et al., 1992). By preventing protein synthesis, DE498 effectively inhibits the growth and development of broadleaf weeds (Frear et al., 1993
). Few studies have been performed regarding toxicity induced by DE498 in mammalian systems.
As a result of the widespread use of AMT and DE498, the lack of toxicity data available on the chemicals, and the occurrence of OA in a plant where there was exposure to these chemicals, the following studies were undertaken to evaluate the potential of AMT or DE498 to induce irritancy, sensitization, and/or systemic toxicity. Further studies investigated the mechanism by which AMT induced sensitization and airway hyperreactivity (AHR).
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MATERIALS AND METHODS |
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Chemicals.
3-Amino-5-mercapto-1,2,4-triazole (AMT; 99% pure; Fig. 1A) was obtained from Acros Organics (NJ) and from the factory where the cases of OA occurred. AMT was tested at concentrations of 1, 5, 10, 15, and 25% (w/v) in dimethyl sulfoxide (DMSO; Sigma, St. Louis, MO), based on limits of solubility. DE498 (factory obtained; Fig. 1B
) was tested at concentrations of 5, 15, 25, and 45% (w/v) in DMSO, based on limits of solubility. Toluene 2,4-diisoyanate (TDI; 99.6% pure) and
-hexylcinnamaldehyde (HCA; 85% pure) were used as positive controls for the Local Lymph Node Assay (LLNA) and phenotypic analysis assays; 2,4-dinitroflourobenzene (DNFB; purity > 99.4%) was used as a positive control for the mouse ear swelling test (MEST). Positive controls were dissolved (w/v for all chemicals) in either DMSO or acetone (99.99% pure). All chemicals used for positive controls or vehicles were obtained from Sigma.
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Twenty-eight day toxicology study.
Prior to the initiation of exposure, BALB/c mice (study 1, n = 10/group; study 2, n = 5/group) were weighed, and the hair was clipped from their dorsal thoraco-lumbar region. Animals were dosed on the clipped site for 28 consecutive days with 50 µl of either vehicle or test chemical. On day 29, animals were weighed, sacrificed by CO2 asphyxiation, and blood was collected by cardiac puncture. In study 1, blood from five animals per group was collected into EDTA tubes for hematology analysis, and blood from the remaining five animals per group was collected into serum separator tubes for chemistry analysis. In the second study, sera were evaluated for serum chemistries, corticosterone, triiodothyronine (T3), and thyroxin (T4) levels. In the second study, to reduce stress which could modulate corticosterone levels, animals were housed individually in covered cages 24 h prior to sacrifice. Mice were sacrificed within one minute of handling the cage, and blood collected promptly after CO2 asphyxiation. In both studies, following blood collection, the animals were necropsied, and organ weights (brain, thymus, lungs, spleen, liver, adrenal glands, and kidneys) were taken. Organ/brain and organ/body ratios were evaluated as indicators of target organ toxicity.
Local Lymph Node Assay (LLNA).
The LLNA was used for an in vivo confirmation of sensitization potential, and was performed according to the protocol recommended by the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM), with minor modifications (ICCVAM, 1999). Briefly, CBA or BALB/c mice (n = 5/group) were dosed with test chemical, vehicle, or positive control on the dorsal surface of each ear for 3 consecutive days. On day 6 mice were intravenously injected with 20 µCi 3H-thymidine (Perkin Elmer, MA). Five h after injection, mice were sacrificed, and a single-cell suspension was prepared from pooled right and left draining lymph nodes (DLNs) of each animal. The uptake of 3H-thymidine was determined using a ß scintillation counter, and a stimulation index (SI) was determined for each experimental group by dividing the group mean by the mean of the vehicle controls. A chemical was classified as positive if at least one concentration reached a SI of
3 with the results being statistically significant and dose-responsive.
Mouse ear swelling test (MEST).
The MEST, used to evaluate contact hypersensitivity inducing potential, followed the procedure described by Hayes et al. (1998), with slight modifications. On days 13 (induction phase), BALB/c mice were dosed on the clipped dorsal thorax with 50 µl of vehicle, test chemical, or positive control. On day 8 the thickness of the right ear pinna of each mouse was measured prior to chemical challenge, using a modified engineers micrometer (Mitutoyo Corporation, Japan). Mice were then challenged with 25 µl of vehicle, test chemical, or positive control chemical on the dorsal surface of the right ear (see Table 1
for dosing concentrations). The thickness of the right ears of all mice were measured 24 and 48 h after challenge, and percentage ear swelling was calculated as follows: ([Mean post-treatment ear thickness / mean pre-treatment ear thickness] x 100) 100. The response of the background control group was compared to the vehicle group as an indicator of the irritancy of the challenge concentration. Test article groups were compared to the background control for significance and dose response, and the positive control was compared to the background positive control (Table 1
).
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Cytokine evaluation.
The dosing protocol for cytokine evaluation consisted of 3 days dermal exposure (as in the LLNA) followed by collection of the DLNs for mRNA analysis 24 h following the final exposure. This time point was chosen based on the studies by Manetz and colleagues (2001), which demonstrated the potential for differentiation of T-cell versus IgE-inducing sensitizers following this dosing regime. Twenty-four h after the last exposure, mice were sacrificed by CO2 asphyxiation, and both DLNs were excised aseptically. Lymph nodes were flash frozen with liquid nitrogen and stored at -80°C. Total RNA was isolated according to Qiagens (Valencia, CA) protocol for total RNA isolation with DNase treatment. Reverse transcription was performed with a TaqMan Reverse Transcription kit in a GeneAmp PCR System 9700 version 1.25 (Applied Biosystems, NJ) according to manufacturers instructions. Copy DNA levels were measured by real-time polymerase chain reaction (RT-PCR) using TaqMan reagents (IL-4, -5, -10, and -12p40, GAPDH, and IFN-
) as prescribed by manufacturers instructions (Applied Biosystems). Biomarkers chosen to evaluate the Th2 response included IL-4, 5, and 10 (Mosmann et al., 1986
), while Th1 biomarkers included IFN-
and IL-12 (Finkelman et al., 1988
; Trinchieri, 1994
).
Data is expressed as fold increase over vehicle, calculated by the following formula: 2Ct, where
Ct =
Ct(Sample)
Ct(Vehicle). The
Ct = Ct(GAPDH) Ct(target), where Ct = cycle threshold as defined by manufactures instructions (Applied Biosystems).
Total serum IgE analysis.
To evaluate the time course of total serum IgE production, BALB/c mice (n = 5/group) were dosed with 25 µl of test article (5, 15, or 25% AMT) or vehicle (DMSO) on the dorsal surface of each ear 5 days a week for 68 days. Mice were tail bled prior to exposure (day 0) to obtain baseline total serum IgE levels, and every two weeks thereafter to monitor alterations in total serum IgE levels.
Total serum IgE was quantified following the procedure described by Manetz and Meade (1999) with minor modifications. A rat
-mouse IgE monoclonal Ab (B1E3) was used as the capture antibody at a concentration of 6 µg/ml. B1E3 hybridomas were generously provided by Daniel Conrad (Virginia Commonwealth University). Samples were applied to the plates at an initial dilution of 1:40 and serially diluted (1:2) down eight wells. A PharMingen (Torreyanna, CA) purified mouse IgE clone IgE-3 (anti-TNP), was used as the standard, at a starting concentration of 5000 ng/ml and serially diluted (1:2) through 12 wells. The secondary antibody, biotinylated anti-mouse IgE, clone R35-92 (PharMingen), was used at a concentration of 2 µg/ml. A 1:400 dilution of streptavidin alkaline phosphatase (SAP, Sigma-Aldrich Cat# S-2890) was used, followed by detection with substrate [p-nitrophenyl phosphate tablets (Sigma-Aldrich) added to substrate buffer]. Plates were read on a Beckman Vmax model plate reader at wavelengths of 405 and 650 nm, and data were analyzed with softmax 3.1.1 ELISA software. Plates were analyzed when the standard reached an OD of at least 1.5, but did not exceed 2.1. Test samples were quantitated by comparison to the standard curve.
Airway hyperreactivity.
In the initial study, BALB/c mice (n = 10/group) were dosed with 25 µl of test article (5, 15, or 25% AMT) or vehicle on the dorsal surface of each ear, 7 days a week, throughout the study. Total serum IgE was evaluated on days 0, 22, and 36. Nonspecific airway hyperreactivity was evaluated on day 21, while specific airway challenge was performed on days 28 and 35. Airway hyperreactivity was assessed in mice as described previously (Howell et al., 2002) with minor modifications. For nonspecific challenge, baseline enhanced pause (PenH) values were obtained in Buxco whole body plethysmography chambers over a 5-min period. Mice were then challenged by nebulizing increasing concentrations (10, 25, and 50 mg/ml) of methacholine (MCH; Sigma) into the chambers. At each concentration of MCH, PenH was assessed over a 5-min period with exposure occurring for the first 3 min. For antigen-specific challenge, mice were lightly anesthetized using isoflourine (Abbott Laboratories, 99.9%) inhalation and exposed by intratracheal (IT) aspiration of 250 µg AMT / 50 µl PBS suspension. Prior to challenge, a 5-min baseline PenH was assessed for each animal. Following challenge, animals were immediately returned to the chambers and PenH was assessed over the following 6 h. Twenty-four h following the final IT challenge, mice were sacrificed by CO2 asphyxiation, and blood was collected following transaction of the abdominal aorta. Lungs were fixed by insufflation of 1 ml 10% buffered formalin via the trachea and placed in 10 ml of 10% buffered formalin for histopathology analysis. Hematoxilin and eosin (H&E), eosinophil (0.5 Chromotrope 2R), mucin (Mayers Mucicarmine), and mast cell staining (Toluene Blue) were performed.
A second study was performed to fill in the dose response curve for AHR. BALB/c mice (n = 15/group) were dosed with 25 µl of test article (5, 10, 15, 20, or 25% AMT) or vehicle on the dorsal surface of each ear, 7 days a week, throughout the study. All animals were tail bled prior to dosing and on day 22 to evaluate total serum IgE levels. On day 35, five animals per group were challenged with MCH as previously described. An additional five animals per group were challenged by intratracheal aspiration with AMT on days 28 and 35. The remaining five animals per group received only dermal exposure. All animals were sacrificed on day 36 (IT challenged animals were sacrificed 24 h post final IT exposure) by CO2 asphyxiation, and blood was collected following transection of the abdominal aorta. Immediately after sacrifice, lungs were insufflated with 10% formalin for histology analysis.
Statistics.
One-way analysis of variance (ANOVA) was performed to analyze variability between at least three experimental groups. If a p value of 0.05 was achieved, Dunnetts posttest was performed to compare test groups to their appropriate control. Differences were considered statistically significant at p < 0.05. To evaluate dose response relationships, linear regression was calculated. The values are considered statistically significant at p < 0.05. When only two groups were analyzed, a t test was performed, with differences considered significant at p < 0.05.
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RESULTS |
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AMT, but Not DE498, Tested Positive for Sensitization in the Local Lymph Node Assay
While efforts are underway to develop sensitive and specific QSAR models for the evaluation of chemical sensitization potential, these have not been accepted as stand-alone methods. Therefore, to confirm the QSAR results, AMT and DE498 were tested in vivo for the potential to induce sensitization using the LLNA at concentrations ranging from 1 to 45%. With factory-obtained AMT, a dose-responsive increase in proliferation was observed (p < 0.05) in CBA mice, with stimulation indices (SI) of 6.20 and 8.86 at the 15% and 25% AMT concentrations, respectively (Fig. 2A). Given that subsequent studies to evaluate the IgE-inducing potential of AMT were to be conducted using BALB/c mice, studies were conducted to compare the LLNA response of this strain with the response in CBA mice. Figure 2B
shows a representative study of two experiments in which a dose-responsive (p < 0.05) increase in lymph node proliferation was induced in BALB/c mice following exposure to AMT. The 15 and 25% dose groups were significantly elevated (p < 0.01) over the DMSO control with SIs of 3.45 and 4.08, respectively, identifying AMT as a sensitizer. In contrast, DE498 tested negative for sensitization potential in the LLNA, as demonstrated by a representative study (one of two studies) where exposure to up to 45% DE498 did not induce an increase in lymph node cell proliferation in BALB/c mice as compared to control animals (Fig. 2C
).
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AMT Was Negative for Contact Hypersensitivity Inducing Potential as Evaluated by the MEST
Given that both IgE and T-cell mediated sensitizers may test positive in the LLNA, as a means to further elucidate the mechanism of sensitization, contact hypersensitivity potential was further evaluated using the MEST. None of the test concentrations induced a significant increase in ear swelling over the BC group at either 24 or 48 h post challenge. The positive control group demonstrated a 49% and 85% increase in ear swelling at 24 and 48 h, respectively. Results are shown in Figure 3.
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DISCUSSION |
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Initial screening using QSAR yielded negative results for irritancy for both AMT and DE498. Earlier studies using the Draize ocular irritancy rabbit test reported slight irritation at 24 h post DE498 challenge with resolution by 48 h (Kleschick et al., 1992). The absence of any indication of irritancy in the TOPKAT evaluation is incongruent, given that the training set for the irritation module for the TOPKAT program is based on Draize test results (Accelyrs, Inc.). No previous evaluation of the irritancy potential of AMT was found in the literature.
Both computer and animal modeling were utilized to characterize the sensitization potential of these chemicals. In the QSAR model proposed by Ashby et al. (1995), chemicals are grouped into categories based on their major reactive subgroups as well as their sensitization potential as evaluated in the LLNA. In this model, chemicals with NH2 reactive groups, classified as electrophiles, tended to test positive in the LLNA, whereas sulphonates with large side groups lost reactive potential due to decreased ability to translocate across the stratum corneum. Based on this classification, one would predict AMT to be positive in this model, as it contains a NH2 side group, while DE498 may be hindered from penetrating the stratum corneum. In another QSAR model, Karol et al. (1996)
associated biological activity with major biophores, or fragments of the molecules statistically associated with sensitization based on the LLNA. The four biophores associated with contact sensitivity include (1) nitrogen-to-carbon double bonds or nitrogen-to-nitrogen double bonds, (2) substituted aromatic structures, (3) sulfur and sulfur-containing moieties, and (4) electrophiles. Based on this QSAR analysis, the 5-mercapto or 3-amino side groups would be potential reactive sites for AMT. In DE498, the 3-mercapto group of AMT is incorporated into a sulfonamide structure, while the 5-amino group of AMT is incorporated into a pyrimidine base. Lacking these reactive moieties, the DE498 was negative for sensitization potential in the TOPKAT QSAR model and the LLNA.
Although originally developed and validated as a method to detect contact sensitizers (ICCVAM, 1999; Kimber et al., 1986
), the LLNA has also been shown to produce positive results following exposure to chemicals that induce an IgE-mediated response. Examples of respiratory sensitizers testing positive in the LLNA include phthalic anhydride (Dearman and Kimber, 1992
), TMA (Dearman and Kimber, 1992
; Manetz and Meade, 1999
), and TDI (Manetz and Meade, 1999
; Woolhiser et al., 1998
). Initial data, which yielded a positive response in TOPKAT QSAR (based on the GPMT) and LLNA, suggested the potential for AMT to induce a DTH reaction. Yet, AMT was negative in the MEST, another murine assay for contact sensitivity. Disparate results seen in the LLNA versus the MEST could in part be due to the phase in which the response was measured. A positive result in the LLNA is based on the initial sensitization phase, while a positive result in the MEST is dependent on the elicitation phase. In a study by Howell et al. (2000)
, the LLNA was able to identify contact sensitivity to potassium dichromate at lower concentrations (0.25%) as compared to the MEST (requiring an induction concentration of 0.5%). Higher induction doses of acrylate compounds were also required to induce a positive response in the MEST as compared to the LLNA (Hayes and Meade, 1999
). Many chemicals have also been shown to elicit both a contact and IgE-mediated hypersensitivity response. Dearman et al. (1992)
demonstrated both a DTH and IgE-mediated hypersensitivity response in BALB/c mice following the dermal application of 50% TMA. Glutaraldehyde has been demonstrated to induce contact dermatitis in both health care workers and workers in the cosmetic industry (Goncalo et al., 1984
; Kiec-Swierczynska and Krecisz, 2001
), while other reports have shown respiratory sensitization and the induction of occupational asthma upon glutaraldehyde exposure (Quirce et al., 1999
; Stenton et al., 1994
).
The potential for AMT to induce sensitization through an IgE-mediated mechanism was supported by local induction of IL-4 mRNA and production of IgE in the DLNs (as measured by an increase in IgE+B220+ lymph node cells). The evaluation of IL-4 mRNA was chosen over the measurement of protein levels, in that PCR is more sensitive than the ELISA, and ConA is not required to induce measurable levels of transcript with PCR, whereas this mitogen has been shown to be required to induce quantifiable levels of IL-4 protein. Results obtained by Ryan et al. (1998) demonstrated similar trends in IL-4 mRNA and protein levels following exposure to TMA, while DNCB, a contact allergen, did not induce increased expression of either IL-4 mRNA or protein. In addition, Warbrick et al. (1998)
demonstrated that TMA induced mRNA and protein levels of IL-4 and IL-10, while DNCB elicited an increase in IFN-
mRNA and protein levels. Further support for the association of increased IL-4 with the development of respiratory sensitizers is provided by Dearman et al. (1996)
, who showed that TMA, cyanuric chloride, and diphenylmethane diisocyanate induce a Th2 cytokine profile, while DNCB, isoeugenol, and formaldehyde induce a Th1 cytokine profile. Manetz and Meade (1999)
demonstrated an increase in IgE+B220+ cells in DLNs following exposure to the IgE-mediated allergens (TDI, TMA), as compared to an increase in B220+ cells in the absence of increased IgE+B220+ cells following exposure to contact allergens. In addition to the local Th2 response, following 26 days of dermal exposure to AMT, an increase in total serum IgE was observed.
The increase in nonspecific AHR to MCH challenge in mice dermally exposed to AMT is consistent with the findings from humans exposed to AMT. From the cluster of OA cases potentially caused by exposure to AMT, of the six cases for whom NIOSH was able to obtain medical records, five tested positive for nonspecific bronchial hyperreactivity. Following removal from AMT exposure, all three of the subjects who were reevaluated by NIOSH no longer had an increase in nonspecific bronchial hyperreactivity (Hnizdo and Sylvain, 2003).
Two observations in these studies are inconsistent with an IgE-mediated mechanism of sensitization. Animals challenged IT with AMT demonstrated a lack of antigen-specific AHR, although the absence of this response could possibly be related to ineffective pulmonary distribution of the chemical following IT administration of a suspension. Additionally, histopathology was inconsistent with an IgE-mediated mechanism, in that there was an absence of eosinophilia and lack of increased mucin present in the lungs.
Taken together, these studies show a lack of dermal sensitization potential for DE498. Computer and animal modeling demonstrate that AMT is not an irritant or systemically toxic at the concentrations tested, yet has the potential to induce sensitization following dermal exposure. Although the mechanism is not yet defined, the development of dermal sensitization and subsequent pulmonary hyperreactivity in BALB/c mice suggests the potential for AMT to induce occupational asthma.
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ACKNOWLEDGMENTS |
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NOTES |
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