MAY 2009 VOLUME 22, NUMBER 5 Copyright 2009 by the American Chemical Society
Letters to the Editor Acrolein: Excessive Cytotoxicity or Potent Mutagenicity? Received March 11, 2009
To the Editor: I am deeply concerned about the publication in Chemical Research in Toxicology of the article by Wang et al. (1). The reported findings in this paper do not support the conclusions drawn by the authors. In this report, Wang et al. (1) have treated the pSP189 plasmid under nonphysiological conditions with excessively high concentrations of acrolein, ranging from 0.1 to 2.5 mM, for prolonged period of time, that is, 16 h at 37 °C at pH 8.0. The authors have conducted LM-PCR footprinting on the supF gene of the chemically modified plasmid and performed the supF mutagenesis assay on the acrolein-treated plasmid transfected into human lung fibroblasts. On the basis of their reported results, Wang et al. (1) have concluded that “Acr-dG adducts are mutagenic and induce G to T and G to A mutations that are similar to the mutational signature found in the p53 gene in the CS-related lung cancer”. They have gone on to hypothesize that “Acr is a major etiological agent for CS-related lung cancer”. I have a number of concerns regarding their data presentation, analysis, and interpretations, which are listed below. 1. The authors state that “Acr-dG adducts induce mutations and that the mutation level is proportional to the amount of Acr used for DNA modifications; the increase of mutations in pSP189 plasmid DNA modified with 0.1, 0.5, 1, and 2.5 mM Acr is 3.5-, 6-, 13-, and 23-fold, respectively” (1). In their earlier publication (2), the authors have reported a 30-fold increase in mutation frequency in the same plasmid modified with 0.1 mM acrolein using the exact same experimental assay “(an increase from 4 × 10-4 in control to 120 × 10-4 in acrolein-treatedplasmid)”. It is unclear how a similar concentration of acrolein could induce such a different mutation frequency in their two seemingly comparable studies (i.e., 8.6-fold lower than the originally reported value). It is also perplexing why a concentration of 2.5 mM acrolein (25 times higher than the originally tested concentration) could not even induce a comparable mutation frequency to that reported earlier. What is more puzzling is that in their recent study, Wang et al. (1) have even
intensified their treatment condition by increasing the acrolein incubation time to 16 h as compared to 6 h used in their earlier report. It is well-established that acrolein is readily and highly reactive toward cellular nucleophiles, such as DNA, without requiring biotransformation to DNA adduct-forming species (3-5). In view of the known reactivity of acrolein with DNA (3-5) and the potent mutagenicity of this chemical in the supF system reported by this same group (2), Wang et al. (1) should not have felt the necessity to increase either the concentration of acrolein or the duration of treatment. If anything, a rational approach would have been to minimize the harshness of treatment by at least not prolonging the incubation time of the naked DNA with acrolein. The latter approach is commonly used to avoid/reduce adventitious DNA damage due to, for example, hydrolytic depurination, oxidation, etc. during in vitro treatment of plasmid DNA. 2. The authors state that “Acr-dG adducts induce mainly G:C to T:A and G:C to A:T mutations” (1). I analyzed their mutation spectrometry data given in Table 2 and found that there is no statistically significant difference in the frequency of the respective types of mutation between acrolein-treated plasmid and control. More specifically, G:C to T:A transversion mutations comprised 48 (45 out of 93) and 43% (6 out of 14) (P ) 0.921; χ2 test), of all base substitutions in acrolein-treated and control plasmid, respectively. In addition, G:C to A:T transition mutations contributed to 29 (27 out of 93) and 50% (7 out of 14) (P ) 0.207; χ2 test) of all base substitutions in acrolein-treated and control plasmid, respectively. Likewise, G:C to T:A transversion mutations comprised 53 (44 out of 83) and 43% (6 out of 14) (P ) 0.68; χ2 test) of all single base substitutions in acrolein-treated and control plasmid, respectively. Additionally, G:C to A:T transition mutations accounted for 30 (25 out of 83) and 50% (7 out of 14) (P ) 0.248; χ2 test) of all single base substitutions in acrolein-treated and control plasmid, respectively (1). Clearly, none of these differences is statistically significant. Of concern is also the limited number of sequenced mutants for establishing the spontaneous and acrolein-induced mutation spectra, that is, only 18 in control and 98 in 1 and 2.5 mM acrolein-treated plasmids,
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combined. Moreover, the small number of colonies screened for mutations, that is, ∼10000 per experimental condition, in the absence of information on how many times each experimental condition was assayed, could have led to overestimation or underestimation of the mutant frequency and/or mutation spectrometry data. Wang et al. (1) should have exercised caution against inferring too much from their limited database. 3. The authors have used excessively high doses of acrolein in a cell-free environment, that is, concentrations of acrolein, ranging from 0.1 to 2.5 mM for 16 h at 37 °C at pH 8.0. The high toxicity of acrolein both in vitro and in vivo is well-documented (3-6). We have recently demonstrated that acrolein treatment of mouse embryonic fibroblasts caused concentration-dependent cytotoxicity, most visibly in the micromolar dose range. At a dose of 0.1 mM acrolein, cell survival was diminished to as low as 7.7%, beyond which cell viability was absolutely obliterated (6). We have now extended our cytotoxicity examination to four normal human cell types, including fibroblasts, keratinocytes, bronchial epithelial cells, and melanocytes. In all cases, acrolein exhibited a similar pattern of cytotoxicity in human cells, whereas 0.1 mM acrolein treatment of all four different cell types resulted in 80-90% cytotoxicity; concentrations of acrolein exceeding 0.5 mM were completely lethal to all of these four normal human cell types. I would like to stress that in our experiments, acrolein treatment did last for 6 h. It is obvious that the nonphysiological doses of acrolein administered by Wang et al. (1) are not attainable in relevant cell culture systems. The excessive doses of acrolein used by these authors did, in fact, generate so many DNA adducts throughout the short sequence of the supF gene (see Figure 6 in ref 1), which makes their discussion on preferential DNA adducts formation and its relationship to the so-called mutational hotspots less relevant. For example, on the transcribed strand, and even more so on the nontranscribed strand of this gene, one can easily see overwhelming DNA adducts formation virtually throughout the sequence; for example, see the indiscriminate formation of DNA adducts on the nontranscribed strand, starting from nucleotide position ∼10 through to ∼70 or perhaps even further away (1). 4. To put the Wang et al. (1) data into prospective, I compared their quantified levels of acrolein-DNA adducts in the chemically modified pSP189 plasmid with the detected levels of respective DNA adducts in human tissues. Using the LC-ESIMS/MS method, Wang et al. (1) have quantitated 155-890 acrolein-DNA adducts (γ- and R- OH-Acr-dG, combined; µmol/mol dG) in the pSP189 plasmid treated with 0.2-1 mM acrolein (see Table 1 in ref 1). Please note that for mutation spectrometry analysis, the authors have used 1 and 2.5 mM acrolein treatment (1). Nath et al. (7) have detected a mean level of 1.36 ( 0.90 acrolein-DNA adducts (µmol/mol G) in oral tissues of smokers using the 32P-postlabeling HPLC technique. Zhang et al. (8) have determined a mean acroleinDNA adduct level of 0.68 ( 0.58/107 nucleotides (γ- and R-OH-Acr-dG, combined) in lung tissues of current or exsmokers using the LC-ESI-MS/MS method. On the basis of these comparisons, one can easily see that the levels of acroleinDNA adducts induced for DNA footprinting and mutagenicity experiments in the Wang et al. (1) study are orders of magnitude higher than those quantitated in human tissues in vivo (7, 8). 5. I would like to reiterate the importance of putting the DNA damaging and mutagenic potentials of any given genotoxin into the context of its cytotoxicity. Clearly, in a cell-free environment such as the supF system, Wang et al. (1) could introduce excessive number of DNA adducts upon extremely harsh
treatment with acrolein. They could then claim a mutagenic potential for the induced DNA adducts, albeit, with no consideration for the proven cytotoxicity of acrolein at the administered doses (6). To investigate DNA-adduct-mediated mutagenicity of genotoxic agents, one should realize that only in viable and replicating cells can the induced DNA adducts be efficiently translated into mutations. In other words, DNA adduct formation, in and of itself, is not sufficient to produce mutagenesis unless the induced DNA lesions can undergo translesion DNA synthesis (TLS), which is only achievable in viable and proliferative cells. In a cell-free environment, such as the supF system, Wang et al. (1) directly exposed the naked DNA of their mutational target gene to excessive concentrations of acrolein, known to be highly cytotoxic (6). The accessibility of acrolein to DNA in a cellular context is much more complicated because acrolein needs to permeate cell and nuclear membranes to reach the target DNA without reacting with other easily accessible targets, for example, glutathione, protein sulfhydryls, thiol-containing enzymes, etc. (3-5). In conclusion, I would like to emphasize that I do not refute the ability of acrolein to form DNA adducts; however, as we have demonstrated previously (6), acrolein at doses sufficient to produce detectable levels of DNA adducts is not mutagenic to mammalian cells. The existing in vivo data have consistently favored an efficient TLS mostly in an error-free manner across both the major and the minor acrolein-DNA adducts (9-13). In vitro, however, lowfidelity TLS has been reported for both of these acrolein-DNA adducts (12-14). The discrepant mutagenicity of acrolein-DNA adducts in vivo and in vitro has been ascribed to a lack of critical accessory factors in vitro that mediate the catalyzing activities of specialized DNA polymerases operating on acrolein-induced DNA adducts in vivo (11, 15). Lastly, on the basis of the evaluation of the International Agency for Research on Cancer, there is inadequate evidence for carcinogenicity of acrolein in humans or animals (16), thus, inferring an etiologic role for acrolein in human lung cancer as stated by Wang et al. (1) is unwarranted. Clearly, there are major limitations in the Wang et al. (1) study, which need to be addressed before these authors can draw any conclusion on the etiologic involvement of acrolein in human lung cancer.
References (1) Wang, H. T., Zhang, S., Hu, Y., and Tang, M. S. (2009) Mutagenicity and sequence specificity of acrolein-DNA adducts. Chem. Res. Toxicol. 22, 511–517. (2) Feng, Z., Hu, W., Hu, Y., and Tang, M. S. (2006) Acrolein is a major cigarette-related lung cancer agent: Preferential binding at p53 mutational hotspots and inhibition of DNA repair. Proc. Natl. Acad. Sci. U.S.A. 103, 15404–15409. (3) Feron, V. J., Til, H. P., de Vrijer, F., Woutersen, R. A., Cassee, F. R., and van Bladeren, P. J. (1991) Aldehydes: Occurrence, carcinogenic potential, mechanism of action and risk assessment. Mutat. Res. 259, 363–385. (4) Ghilarducci, D. P., and Tjeerdema, R. S. (1995) Fate and effects of acrolein. ReV. EnViron. Contam. Toxicol. 144, 95–146. (5) Kehrer, J. P., and Biswal, S. S. (2000) The molecular effects of acrolein. Toxicol. Sci. 57, 6–15. (6) Kim, S. I., Pfeifer, G. P., and Besaratinia, A. (2007) Lack of mutagenicity of acrolein-induced DNA adducts in mouse and human cells. Cancer Res. 67, 11640–11167. (7) Nath, R. G., Chen, H. J., Nishikawa, A., Young-Sciame, R., and Chung, F. L. (1994) A 32P-postlabeling method for simultaneous detection and quantification of exocyclic etheno and propano adducts in DNA. Carcinogenesis 15, 979–984. (8) Zhang, S., Villalta, P. W., Wang, M., and Hecht, S. S. (2007) Detection and quantitation of acrolein-derived 1, N2-propanodeoxyguanosine adducts in human lung by liquid chromatography-electrospray ionization-tandem mass spectrometry. Chem. Res. Toxicol. 20, 565–571.
Chem. Res. Toxicol., Vol. 22, No. 5, 2009 753 (9) Yang, I. Y., Hossain, M., Miller, H., Khullar, S., Johnson, F., Grollman, A., and Moriya, M. (2001) Responses to the major acrolein-derived deoxyguanosine adduct in Escherichia coli. J. Biol. Chem. 276, 9071– 9076. (10) Yang, I. Y., Johnson, F., Grollman, A. P., and Moriya, M. (2002) Genotoxic mechanism for the major acrolein-derived deoxyguanosine adduct in human cells. Chem. Res. Toxicol. 15, 160–164. (11) Yang, I. Y., Chan, G., Miller, H., Huang, Y., Torres, M. C., Johnson, F., and Moriya, M. (2002) Mutagenesis by acrolein-derived propanodeoxyguanosine adducts in human cells. Biochemistry 41, 13826– 13832. (12) Kanuri, M., Minko, I. G., Nechev, L. V., Harris, T. M., Harris, C. M., and Lloyd, R. S. (2002) Error prone translesion synthesis past gammahydroxypropano deoxyguanosine, the primary acrolein-derived adduct in mammalian cells. J. Biol. Chem. 277, 18257–18265. (13) Sanchez, A. M., Minko, I. G., Kurtz, A. J., Kanuri, M., Moriya, M., and Lloyd, R. S. (2003) Comparative evaluation of the bioreactivity and mutagenic spectra of acrolein-derived alpha-HOPdG and gammaHOPdG regioisomeric deoxyguanosine adducts. Chem. Res. Toxicol. 16, 1019–1028. (14) VanderVeen, L. A., Hashim, M. F., Nechev, L. V., Harris, T. M., Harris, C. M., and Marnett, L. J. (2001) Evaluation of the mutagenic potential of the principal DNA adduct of acrolein. J. Biol. Chem. 276, 9066–9070. (15) Yang, I. Y., Miller, H., Wang, Z., Frank, E. G., Ohmori, H., Hanaoka, F., and Moriya, M. (2003) Mammalian translesion DNA synthesis across an acrolein-derived deoxyguanosine adduct. Participation of DNA polymerase eta in error-prone synthesis in human cells. J. Biol. Chem. 278, 13989–13994. (16) IARC (1995) Acrolein. IARC Monogr. EVal. Carcinog. Risks Hum. 63, 337-372.
Ahmad Besaratinia Division of Biology Beckman Research Institute of the City of Hope National Medical Center 1450 East Duarte Road Duarte, California 91010 Tel: 626-359 8111 ext. 65918 Fax: 626-358 7703 E-mail:
[email protected] TX900098U
Acrolein-DNA Adducts Are Mutagenic Received March 27, 2009
To the Editor: This letter is our response to Dr. Ahmad Besaratinia’s letter. Two major points in Dr. Besaratinia’s letter are as follows: (1) Is the acrolein-deoxyguanosine DNA adduct (Acr-dG) mutagenic? (2) Is Acr an etiological agent of lung cancer? The evidence that Acr-DNA modifications produce two isomeric Acr-dG has been well-established in many laboratories (1-3). Acr modifications in the shuttle vector pSP189 induce targeted G to T and G to A mutations in supF gene; this was first reported by Dr. Kawanishi et al. in 1998 (4). Using UvrABC and ligation-mediated PCR to map Acr-dG distribution in the p53 gene in normal human bronchial epithelial (NHBE) cell and normal human lung fibroblast (NHLF), we have found that the distribution of Acr-dG adducts in the p53 gene coincides with the p53 mutational pattern in the lung cancer of cigarette smokers. We have confirmed that Acr-modified pSP189 indeed induces G to T and A mutations. We have also found that Acr treatment causes an inhibitory effect on nucleotide excision repair (NER). Because Acr is 1000-10000-fold higher than benzo(a)pyrene in cigarette smoke, particularly in side stream smoke, and rich in cooking fumes, we have concluded that Acr is a major etiological agent of lung cancer (5). Ahmad Besaratinia with Kim and Pfeifer published a paper in Cancer Research, among other things, claiming that Acrtreated pSP189 plasmids do not produce mutations (6). Unfor-
tunately, Dr. Besaratinia et al. did not identify and quantify the Acr-dG formation in their DNA modifications. There are two possibilities to account for Dr. Besaratinia et al.’s results: (1) Acr-dG is indeed nonmutagenic, which implies that both ours and Dr. Kawanashi’s results are incorrect, and (2) the Acr-DNA modification conditions that Dr. Besaratinia et al. employed did not produce enough Acr-dG in the pSP189 to manifest Acr mutagenicity. Our current paper (7) was designed to set this record straight. It appears that Dr. Besaratinia misread our paper in two aspects: (1) Acr-dG adducts distribution in the supF gene was determined by UvrABC incision in Acr-modified pSP189 and was not by LMPCR, which amplifies the UvrABC incision signal 106-107-fold, and (2) Acr-dG per supF fragments are 0.02 and 0.04 (1-2 × 85/4952) in Acr-dG modifications using 1 and 2.5 mM Acr under our modification conditions that produce 1-2 Acr-dG per plasmid, if we assume that the modifications are random. This level of Acr modification is not “many and heavily” in this fragment as “interpreted” by Besaratinia. 1. It was found more than 20 years ago that Acr treatment induces mutations in human repair-deficient (XPA) cells but not in repair-proficient normal fibroblasts (NF), even though Acr induces cytotoxicity in both types of cells (8). These results indicate that the major cytotoxicity effect of Acr in NF probably results from damage other than genomic DNA. On the other hand, these results also indicate that Acr-dG can cause mutations at so-called “physiological conditions” in XPA cells. Acr-dG mutagenicity was unambiguous as addressed by Kawanishi et al. (4) using an Acr-modified pSP189 system without the complications of cytotoxicity. By modification of pSP189 with Acr in 0.1 × TE, pH 8.0, at 37 °C for 16 h, we (7) have found that Acr-DNA modifications indeed induce G to T and A targeted mutations similar to Dr. Kawanishi et al.’s findings except that we used a 10-12 times lower concentration of Acr to have the same mutation frequency that Dr. Kawanishi et al. used (4). Several factors can affect the efficiency of Acr-dG formation in in vitro Acr-DNA modifications, such as quality of Acr, buffer solution, pH, temperature, and reaction time. Both ours and Kawanishi et al.’s pSP189 plasmids that produce the same mutant frequency probably have the same level of Acr-dG per plasmid even though different concentrations of Acr were used by them and us. Our results in the current paper show that not only the Acr-dG formation is proportional to Acr concentrations but also the mutation frequency is also proportional to the Acr concentrations. Besaratinia’s concern of how reproducible Acr-DNA modifications are under different conditions is unnecessary and neglects the crucial findings in these experiments, that is, that Acr-dG is mutagenic. 2. Acr modifications induced G to T and A mutations, which have been reported by Kawanishi et al. (4). To address whether these mutations are indeed induced by Acr modifications, we have sequenced the mutants induced by Acr modifications that produce 10-20 times more mutants than without Acr modifications. We have found that the sequence positions of Acr modification-induced mutations are significantly different from the mutation sequence positions that are detected in spontaneous mutants. We sequenced 93 mutants identified from a total of ∼40000 colonies. The number is statistically significant to ensure that the mutations indeed are a result from Acr modifications. Because the nature of spontaneous mutagenesis is unclear, it is meaning-
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less to compare the types of mutations induced by Acr modifications. 3. Dr. Besaratinia failed to understand that the issue of Acr cytotoxicity and the mutagenicity of Acr-dG can be addressed separately using a shuttle vector system commonly used by many laboratories including Dr. Pfeifer’s. In vitro Acr-DNA modification conditions are simply to achieve a reasonable level of Acr-dG per plasmid while maintaining the integrity of the other aspects of the DNA; therefore, AcrdG-induced mutation can be detected. These chemical modification conditions can hardly be the same as physiological conditions. Dr. Besaratinia also misinterprets the Acr-dG distribution results shown in Figure 6 in our paper (7). By virtue of the methodology, only one adduct per supF fragment is detected (remember that there is only 0.02-0.04 Acr-dG adduct/supF fragment); the many bands shown in the gel are accumulative representations of millions of supF DNA fragments! Furthermore, the purpose of identifying the sequence of Acr-dG formation is to compare the mutational spectrum induced by Acr modifications and to demonstrate that the mutations detected are targeted mutations as we expected. 4. Dr. Besaratinia put forth a comparison that Acr-dG detected in smokers’ oral tissue or lung tissue is ∼10-6 /dG to 10-7/ nucleotide, and in contrast, the Acr-dG level in the pSP189 that we used for mutation detection is 1-2/plasmid. This comparison is irrelevant and futile in terms of addressing the mutagenicity of Acr-dG. To address whether Acr-dG is mutagenic or not, Acr-dG has to be formed in the pSP189; if we use pSP189 with 10-fold less Acr-dG adducts, then simply we have to screen 10 times more colonies (∼106) to obtain the same number of mutants for sequencing. Besaratinia probably is aware that it is one Acr-dG per plasmid in the commonly used constructs of plasmids containing a site-directed Acr-dG to determine the mutagenicity. 5. Several factors can affect the efficiency of Acr-dG formation in in vitro Acr-DNA modifications as we addressed above. Acr may react with cellular components better than other aldehydes, but it is slow as compared to diol epoxide forms of polycyclic aromatic hydrocarbons. One necessary thing to ensure proper modifications is to determine the Acr-dG formation by chemical analysis and by the UvrABC incision method. It is rather nonsensical and sentimental for Besaratinia to claim that the conditions that we used (water or 0.1 × TE for 16 h) are too “harsh” in comparison to his 6 h treatment. TE (Tris-EDTA) buffer at pH 8.0 can prevent depurination and oxidation; therefore, DNA in TE buffer is stable. Indeed, we found that pSP189 DNA under our mock Acr-DNA modification conditions does not have an increased supF mutation, and no Acr-dG adducts were detected (5, 7). Furthermore, only 0.02-0.04 Acr-dG /supF is formed in the 1 and 2.5 mM Acr-modified pSP189 under our modification
conditions; this level of Acr-dG/supF is not “many and heavily” as Besaratinia miscalculated. Finally, it has long been established that acrolein is the major toxic metabolite of chemotherapeutic agents cyclophosphamide and ifosfamide, and it has long been suspected that Acr causes secondary bladder cancer (9-11). Acr has been shown to induce bladder cancer in animal models, although its complete carcinogenicity has yet to be demonstrated (12). We do agree with Besaratinia that based on IARC deliberations, there is inadequate evidence to address the role of acrolein in lung cancer in animal models.
References (1) Chung, F. L., Young, R., and Hecht, S. S. (1984) Formation of cyclic 1,N2-propanodeoxyguanosine adducts in DNA upon reaction with acrolein or crotonaldehyde. Cancer Res. 44, 990–995. (2) Stevens, J. F., and Maier, C. S. (2008) Acrolein: Sources, metabolism, and biomolecular interactions relevant to human health and disease. Mol. Nutr. Food Res. 52, 7–25. (3) Zhang, S., Villalta, P. W., Wang, M., and Hecht, S. S. (2007) Detection and quantitation of acrolein-derived 1,N2-propanodeoxyguanosine adducts in human lung by liquid chromatography-electrospray ionization-tandem mass spectrometry. Chem. Res. Toxicol. 20, 565–71. (4) Kawanishi, M., Matsuda, T., Nakayama, A., Takebe, H., Matsui, S., and Yagi, T. (1998) Molecular analysis of mutations induced by acrolein in human fibroblast cells using supF shuttle vector plasmids. Mutat. Res. 417, 65–73. (5) Feng, Z., Hu, W., Hu, Y., and Tang, M. S. (2006) Acrolein is a major cigarette-related lung cancer agent: Preferential binding at p53 mutational hotspots and inhibition of DNA repair. Proc. Natl. Acad. Sci. U.S.A. 103, 15404–15409. (6) Kim, S. I., Pfeifer, G. P., and Besaratinia, A. (2007) Lack of mutagenicity of acrolein-induced DNA adducts in mouse and human cells. Cancer Res. 67, 11640–11647. (7) Wang, H. T., Zhang, S., Hu, Y., and Tang, M. S. (2009) Mutagenicity and sequence specificity of acrolein-DNA adducts. Chem. Res. Toxicol. 22, 511–517. (8) Curren, R. D., Yang, L. L., Conklin, P. M., Grafstrom, R. C., and Harris, C. C. (1988) Mutagenesis of xeroderma pigmentosum fibroblasts by acrolein. Mutat. Res. 209, 17–22. (9) Comes R. M. M., and Eggleton, M. (2002) Concise International Chemical Assessment Document No. 43, World Health Organization, Geneva. (10) Ghilarducci, D. P., and Tjeerdema, R. S. (1995) Fate and effects of acrolein. ReV. EnViron. Contam. Toxicol. 144, 95–146. (11) Kehrer, J. P., and Biswal, S. S. (2000) The molecular effects of acrolein. Toxicol. Sci. 57, 6–15. (12) Cohen, S. M., Garland, E. M., St. John, M., Okamura, T., and Smith, R. A. (1992) Acrolein initiates rat urinary bladder carcinogenesis. Cancer Res. 52, 3577–3581.
Hsiang-tsui Wang Moon-shong Tang* Department of Environmental Medicine Pathology and Medicine New York University School of Medicine 57 Old Forge Road Tuxedo, New York 10987 TX900116W
