1 15 3 https://www.nal.usda.gov/exhibits/speccoll/files/original/a805432627673c28e070d4ebbb50e78d.pdf 32f7d8d61eeaaf541902521464a3a73c PDF Text Text Item D Number 02233 Author Gierthy, John F. New York State Department of Health, Center for Labor Report/Article fitto Typescript: Progress Report: Evaluation of In Vitro Assays for the Detection of "Dioxin-Like" Activity in the Binghamton State Office Building, May, 1983 Journal/Book Title Year 1983 Month/Day Ma 23 Color Number of Images v D 9 Descrlpton Notes Thursday, September 20, 2001 Page 2233 of 2293 �Progress Report: Evaluation of In Vitro Assays for the Detection of "Dioxin-Like" Activity in the Binghamton State Office Building by John F. Gierthy and Gerald D. Frenkel New York State Department of Health Center for Laboratories and Research May, 1983 RECEIVED MAY 2 3 1983 DIRECTOR PUBLIC HEALTH �Progress Report: Evaluation of In Vitro Assays for the Detection of "Dioxin-Like" Activity.in the Binghamton State Office Building. Gierthy, J.F. and Frenkel, G.D. (May, 1983) Previous reports have described the development and application of two in vitro systems for the detection of "dioxin-like" activity in extracts of soot contaminated with polychlorinated dibenzodioxins and dibenzofurans. The cell keratinization system, based on the induction of keratinization in epithelial cells by exposure to 2,3,7,8-TCDD, and the flat cell assay, based on a 2,3,7,8-TCDD induced change in cellular morphology have both been shown to be potentially useful in the detection of dioxin-like activity in extracts of soot from the Binghamton State Office Building (BSOB). Further verification of the specificity of the flat cell induction to 2,3,7,8-TCDD exposure has been carried out. Various polychlorinated dioxins (PCDDs) and dibenzofurans (PCDFs), polychlorinated biphenyls (PCBs), polynuclear aromatic hydrocarbons (PAHs), and pesticides have been tested for their ability to induce the flat cell morphology. Results show that 2,3,7,8-TCDD is the most potent inducer of this morphological change, while the other PCDD and PCDFs have activity ranging from 10 to 10 of this isomer. The PCBsi,PAHs and pesticides had lower activity ranging from 10 to less than 10 that of 2,3,7,8-TCDD. Where comparison was possible, a good correlation was seen between the published relative activity of compounds in the keratinization system and their activity in the flat cell assay. These results suggest that the sensitivity and specificity of the flat cell assay is similar to that of the keratinization system. Previously, benzene extracts of cotton gauze swipes from the BSOB were found to contain a substance which precipitated during solvent exchange to DMSO and resulted in a loss of activity in the bioassays. This substance has now been found to be associated with the cotton gauze swipe pads. Results show that pre-extraction of the gauze pads with benzene allows full recovery of activity. �1 Hyperkeratinization is considered to be responsible for the occurrence of chloracne in humans exposed to polychlorinated dioxins and dibenzofurans (Poland et al_., 1982). This effect is thought to be caused by an induced differentiation of the squamous epithelium. In this regard, 2,3,7,8-tetrachlorodibenzo(p)dioxin (2,3,7,8-TCDD) is considered to produce a sustained stimulation of a normal physiological response which leads to chloracne (Poland e_t aK, 1982). Knutson and Poland have used the in vitro XB/3T3 cell keratinization system (Rheinwald £t ai., 1975) to study this effect of chlorinated dioxin isomers and congeners (Knutson et al., 1980). In previous reports (Gierthy e£ al., 1982a,b) we have demonstrated the use of this system as an assay for the detection of these compounds in Binghamton State Office Building (BSOB) soot extracts. During these experiments, the appearance of an altered cell morphology was observed in those cells which had been exposed to 2,3,7,8-TCDD or the soot extracts (Gierthy £t al., 1982a,b). This change was first seen after 7 days of exposure to the samples and was characterized by a flat cell morphology as compared to the more fusiform cells in the unexposed —11 cultures or those exposed to less than 10 M 2,3,7,8-TCDD. An apparent cessation in cell growth was also seen in these cells, while the unexposed cells continued to proliferate, resulting in more dense cultures. The minimal concentration of each soot sample extract capable of inducing the flat cell response was recorded as a measure of relative activity. The correlation between the concentration required for induction of the keratinization response and the flat cell morphology (Gierthy et_ al., 1982a) indicated that it may be possible to use the flat cell morphological change in these cells as an alternative and improved endpoint in the assay for dioxin-like activity. �The reproducibility of the assay as well as its ability to differentiate between various concentrations of 2,3,7,8-TCDD has been shown to be comparable to that of the cell keratinization system (Gierthy et al., 1982b). Other cell lines, including fibroblasts and transformed epithelial cells, were tested for their ability to respond to 2,3,7,8-TCDD with a change in morphology, but no flat cell effect was observed. Various inhibi- tors of macromolecular synthesis and mitosis were examined for their ability to induce flat cell morphological change. As in the keratinization system (Knutson et al^., 1980), all were found to be inactive over a broad range of concentrations (Gierthy et^ a_^., 1982b), indicating that the effect is not associated with general toxicity but may be a specific effect, analogous to, and perhaps related to, keratinization. This possi- bility was supported by the finding that 2,3,7,8-tetrachlorodibenzofuran (2,3,7,8-TCDF) also induced the flat cell effect (Gierthy et al., 1982b), but had approximately one tenth the potency as 2,3,7,8-TCDD, which is the relative potency which has been reported for these two compounds in the induction of keratinization (Knutson et_ al_., 1980). This report describes further experiments which have been performed to establish the validity of the flat cell system as an assay for dioxin-like activity, as well as preliminary studies regarding the application of the in vitro assays to the detection of dioxin-like activity in surface swipe samples taken from the BSOB. Results and Discussion Further Validation of the Flat Cell Assay including polychlorinated dibenzo(p)dioxins Studies on 24 chemicals, (PCDDs), polychlorinated dibenzofurans (PCDFs), polychlorinated biphenyls (PCBs), polycyclic aromatic hydrocarbons, and pesticides have indicated at least a 63 million �fold range in the potential of these compounds for inducing the flat cell effect (Table 1). These data show that the flat cell effect, like the keratinization reponse, is by far, most sensitive to the more toxic PCDDs and PCDFs with 2,3,7,8-TCDD being the most potent. Specifically, 1,2,4,7,8-penta-CDD, which lacks chlorination in one of the lateral positions of one of the benzene rings shows 100 fold less activity than 2,3,7,8-TCDD. 2,3,7,8-TCDF was shown to be the most potent PCDF tested and, as in the keratinization system (Knutson et^ al., 1980), had a flat cell inducing activity an order of magnitude lower than 2,3,7,8-TCDD. Other PCDFs tested gave a range of activity, with the hexa-CDF being more potent than the octa- or di-CDFs. The range of activity observed for the PCDDs and PCDFs tested, relative to 2,3,7,8-TCDD was about 1,000 fold. This is contrasted by the activity of the various PCBs tested. Here the flat cell inducing activity was 10 -10 2,3,7,8-TCDD. times less potent than Similar relative activity was reported in the keratinization system for another halogenated biphenyl, 2,3,4,2',3',4'-hexabromobiphenyl (Knutson e£ al., 1980). The polynuclear aromatic hydrocarbons varied in activity in relation to 2,3,7,8-TCDD. benz[ajanthracene were about 10 2,3,7,8-TCDD respectively. 3 Dibenzo[a,h]anthracene and 4 and 10 times less potent than This correlates well with the reported activities of these compounds in the cell keratinization system (Knutson et^ al^., 1980). 3-Methylcholanthrene and benzo(a)pyrene were both toxic at concentrations which were insufficient to induce a significant flat cell effect. These compounds were found to be inactive as inducers of keratinization in the XB/3T3 system (Knutson £t £1^., 1980). The pesticides were all inactive in inducing the flat cell response at the concentrations tested with the exception of Mirex which had the highest �potency of this group (10 times less active than 2,3,7,8-TCDD). These data indicate that the specificity of the flat-cell assay appears similar to that of the cell keratinization system. Application to BSOB Samples Versar New York Inc. (Springfield, Va.) has supplied twenty-nine benzene extracts of soot samples from the BSOB for preliminary testing of the bioassays. These included benzene extracts of cotton gauze swipes of BSOB surfaces, matrix blanks and reagent blanks. Upon solvent exchange into DMSO, a precipitate was formed in all of these samples except the reagent blank and therefore seemed to be associated with the gauze pad extracts. Furthermore when the matrix blanks and reagent blanks were spiked with 2,3,7,8-TCDD prior to solvent exchange, the activity of spiked matrix blank samples was significantly lower than that of spiked reagent blanks. The presence of a waxy precipitate was confirmed by similar solvent exchange performed by Versar on the cotton gauze swipes (Sonchik, 1982). Versar also noted that a PCS spike (Aroclor 1254, 1 ug/pad) partitioned into the precipitate, thus reducing the concentration found in the DMSO. Versar subsequently supplied benzene extracts of gauze pads which had been pre-extracted with benzene. When these samples were solvent exchanged as before no precipitate was observed in three samples and only a very slight precipitate in one of the samples. Furthermore when these samples were spiked with 2,3,7,8-TCDD prior to solvent exchange, full bioassay activity was recovered. No bioassay activity was found when unspiked samples were tested. From these results, it can be concluded that the precipitate which formed during solvent exchange of the benzene extract of the gauze swipes was a substance which had been extracted from the gauze. This precipitate �prevented full recovery of added 2,3,7,8-TCDD (as well as PCBs (Sonchik, 1982)). Pre-extraction of the cotton gauze pads with benzene effectively removes this substance. It appears that this procedure will allow further studies to be done concerning the feasibility of using the in vitro bioassays to detect dioxin-like activity in the surface swipe samples from the BSOB. �Table 1 Induction of the Flat Cell Effect by Various Chemicals Minimum Detectable Concentration (ppb) 2,3, 7,8-Tetrachlorodibenzo(p)dioxin 0.003Z -=• ~\ l,2,4,7,8-Pentachlorodibenzo(p)dioxin ro.359j> 2,3, 7,8-Tetrachlorodibenzofuran 0.032 2,3,4,6,7, 8-Hexachlorodibenzof uran 0.378 Octachlorodibenzofuran 4.48 2,6-Dichlorodibenzofuran >2.38 3,4,3' ,4'-Tetrachlorobiphenyl 100 2,4,5,2' ,4' ,5'-Hexachlorobiphenyl 1,000 2,5,2' ,5'-Tetrachlorobiphenyl >10,000 2,3,4,2' ,4' ,5'-Hexachlorobiphenyl >10,000 2,3,4,2' ,3' ,4'-Hexachlorobiphenyl >10,000 Aroclor 1254 10,000 Dibenzo [a,h ]anthracene 10 Benz [a ]anthracene 100 >iooa >iooa 3-Methylcholanthrene Benzo(a)pyrene 6-Naphthoflavone 1,000 Pyrene >10,000 Mirex 10,000 Dieldrin >10,000 Aldrin >10,000 o.p'DDT >10,000 Lindane >10,000 a -BHC >200,000 a Toxic concentration was 1,000 ppb. �Literature Cited Gierthy, J.F. and G.D. Frenkel (1982a) A Preliminary Report "on the Evaluation of an In Vitro Assay for the Detection of "Dioxin-Like" Activity Using Extracts of Soot from the Bingharaton State Office Building. New York State Department of Health Report. Gierthy, J.F. and G.D. Frenkel (1982b) Progress Report: March, 1982. Evaluation of an In Vitro Assay for the Detection of "Dioxin-Like" Activity in the Binghamton State Office Building. Health Report. New York State Department of October, 1982. Knutson, J.C. and Poland, A. (1980). Keratinization of mouse teratoma cell line XB produced by 2,3,7,8-tetrachlorodibenzo-p-dioxin: an in vitro model of toxicity. Cell 22, 27-36. Poland, A. and Knutson, J. (1982) 2,3,7,8-tetrachlorodibenzo-p-dioxin and related halogenated aromatic hydrocarbons: Examination of the mechanism of toxicity. Ann. Rev. Pharmacol. Toxicol. 22, 517-554. Rheinwald, J.G. and Green, H. (1975). Formation of a keratinization epithelium in culture by a cloned cell line derived from a teratoma. Cell 6, 317-330. Sonchik, S. (1982). Analytical Report: Removal of Interferences of Samples for Cell Keratinization Assay. 1982. Versar Report, December 8, � Dublin Core The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/. Title A name given to the resource Alvin L. Young Collection on Agent Orange Description An account of the resource <p style="margin-top: -1em; line-height: 1.2em;">The Alvin L. Young Collection on Agent Orange comprises 120 linear feet and spans the late 1800s to 2005; however, the bulk of the coverage is from the 1960s to the 1980s and there are many undated items. The collection was donated to Special Collections of the National Agricultural Library in 1985 by Dr. Alvin L. Young (1942- ). Dr. Young developed the collection as he conducted extensive research on the military defoliant Agent Orange. The collection is in good condition and includes letters, memoranda, books, reports, press releases, journal and newspaper clippings, field logs and notebooks, newsletters, maps, booklets and pamphlets, photographs, memorabilia, and audiotapes of an interview with Dr. Young.</p> <p>For more about this collection, <a href="/exhibits/speccoll/exhibits/show/alvin-l--young-collection-on-a">view the Agent Orange Exhibit.</a></p> Text A resource consisting primarily of words for reading. Examples include books, letters, dissertations, poems, newspapers, articles, archives of mailing lists. Note that facsimiles or images of texts are still of the genre Text. Box The box containing the original item. 088 Folder The folder containing the original item. 2233 Series The series number of the original item. Series IV Subseries II Dublin Core The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/. Creator An entity primarily responsible for making the resource Gierthy, John F. Gerald D. Frenkel Description An account of the resource <strong>Corporate Author: </strong>New York State Department of Health, Center for Laboratories and Research Date A point or period of time associated with an event in the lifecycle of the resource May 23 1983 Title A name given to the resource Typescript: Progress Report: Evaluation of In Vitro Assays for the Detection of "Dioxin-Like" Activity in the Binghamton State Office Building, May, 1983 Subject The topic of the resource BSOB chemical properties data analysis https://www.nal.usda.gov/exhibits/speccoll/files/original/d18c92ba1c7b5858ce473a3255575d2e.pdf fc5119254c705b4269445699dfd21b7f PDF Text Text Item D Number °2212 Author Gierthy, John F. Corporate Author RODOrt/APtlClO TItiB Typescript: A Preliminary Report on the Evaluation of an In Vitro Assay for the Detection of "Dioxin-Like" Activity Using Extracts of Soot from the Binghamton State Office Building, 1982 Journal/Book Title Year Month/Day Color Number of Images D 28 Thursday, September 20, 2001 Page 2212 of 2293 �A Preliminary Report on the Evaluation of an In Vitro Assay for the Detection of "Dioxin-Like" Activity Using Extracts of Soot from the Binghamton State Office Building ~? "by John F. Gierthy and Gerald D. Frenkel New York State Department of Health Division of Laboratories and Research �.V " ' Abstract. As a result of the involvement of an electrical transformer containing polychlorinated biphenyls (PCBs) and chlorinated "benzenes in a fire in the Binghamton State Office Building (BSOB), the structure was contaminated vith soot ladened with dioxin congeners and isomers, which included 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8 TCDD) and 2,3,7,8-tetrachlorodibenzofuran (2,3,7,8 TCDF). A panel of experts, which was convened to examine decontamination procedurestrecommended the development of a short term assay for these compounds, based on an in vitro model of the in vivo chloracne response to dioxin exposure. This assay uses the dioxininduced keratinization of epithelial cell cultures as an endpoint. This endpoint is evaluated by eye, using intensity of the keratin specific stain, Rhodamine B, as a marker. This report describes the evaluation of the potential utility of such an assay as a rapid, semiquantitative screen for monitoring the BSOB decontamination process. Initial experiments using a 2,3,7,8 TCDD standard established that the kera.tinization model could be duplicated in this laboratory. A soot sample from the building for which levels of 2,3,7,8 TCDD and 2,3,7,8 TCDF had been determined by mass spectrometry, was tested in this in vitro system. The resulting activity was of the same order of magnitude as expected from the content of TCDD and TCDF as determined in the mass spectrometry analysis. Twelve extracts of soot from various floors of the building were tested for their activity in the assay. Activity of soot extracts from the various floors ranged over three orders of magnitude with samples from the lower floors generally having lower activity. These studies have also lead to the observation that after exposure to 2,3,7,8 TCDD, cells in the assay undergo a change in morphology characterized by the appearance of flat, apparently nonproliferating cells. This change in morphology, when used as an endpoint, correlated well with the keratinization endpoint and thus �ii may form the basis of an alternative and possibly better in vitro assay for dioxin congeners and isomers. A possible correlation between the keratinization activity and the PCB content of these soot extracts was observed. Preliminary assays on a PCB mixture (Aroclor 125H), which utilized the flat cell morphology as an endpoint, have indicated that this correlation was probably not due to a direct effect of the PCBs on this assay. The effect of a proposed decontamination agent, Triton X-100, was also tested using this endpoint. A concentration of 0.1 vil/ml was toxic while lower levels had no activity in this assay, nor did they affect the sensitivity of the assay to 2,3,7,8 TCDD. The keratinization assay exhibited a decrease,with time, in the magnitude of the response to the point that keratinization was characterized by the appearance of colonies of keratinizing cells on a background of nonkeratinizing cells. These cultures could be evaluated microscopically and there was no loss of sensitivity to 2,3S7»8 TCDD associated with the decline of the response magnitude. Efforts to determine the cause of the decline, and to reverse it, are discussed. �The occurrence of polychlorinated di"benzo-p-dioxins (PCDD) and dibenzofurans (PCDP) in the environment poses a serious potential threat to human health. These compounds are among the most toxic and teratogenic low molecular weight compounds known (Poland et_ al_. , 1976). • Chlorinated dibenzo-p-dioxins are formed from the condensation of two orthochlorophenates under conditions of high alkalinity, pressure and temperature. The combustion, chemical nature and combustion chamber residence time can also lead to the production of PCDDs and PCDFs. This has been verified by the analysis of flue gas emission and fly ash of some municipal incinerators in the Netherlands (Hutzinger et_ al_. , 198l). (Langer et_ al_. , 1973). This process has also been observed in laboratory models The pyrolysis of organohalogens such as chlorinated phenols and PCBs lead to the formation of PCDDs and PCDPs. This observation was evidenced under field conditions recently in New York State. On February 5, 1983., as a result of fire, the Binghamton State Office Building (BSOB) was heavily contaminated with soot; subsequent cleanup efforts were halted when chemical analysis indicated the presence of high levels of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and 2,3,7,8tetrachlorodibenzofuran (TCDF) as well as other possibly toxic congeners (Smith et al., 198l). This contamination was traced to the involvement by fire of an electrical transformer which contained the dielectric fluid Pyranol, a mixture of PCBs composed of Aroclor 125^ and chlorinated benzenes. A number of human exposures to dioxin congeners and isomers have been reported. An acne outbreak in Germany in 195^ was traced to trichlorophenol con- taminated with TCDD and TCDF (.Poland et_ aJU , 1976). Similar outbreaks of chloracne, the most common manifestation of dioxin exposure in humans, were noted in 2,^,5-T production plants in France, Germany and the United States in the 1950"s and 1960's. In 1971 the use of waste oil contaminated with TCDD for dust control in a riding arena lead to the death of a large number of horses and other small animals, as well as some cases of chloracne in children who played in the area (Kimbrough _et_ al_. , 1977). The contamination of rice oil with dibenzofuran analogs lead to many cases of chloracne in Japan (Bradlaw et al., 1979). The explosion of a chemical plant in Seveso, Italy �2 in 1976 lead to exposure of the population to TCDD which was thought to be responsible for the occurrence of chloracne in humans and a number of livestock losses (Hay, 1980; Pocchiari et_ al_. , 1979; Reggiani, 1978). The use of Agent Orange ( a mixture of n-butyl esters of 2,^-D and 2,^,5-T), a defolient, in Vietnam between 1962 and 1969 lead to the contamination of large areas by TCDD (.Poland et_ al_. , 1976). Recent work by Knutson and Poland has demonstrated that the hyperkeratinization response to TCDD exposure which occurs upon exposure to TCDD in vivo could be reproduced in an in vitro model system (Knutson et_ al_. , 1980). The induction of keratinization is thought to be responsible for the in vivo induction of chloracne. They found that although TCDD did not produce an acute toxic response in various cell types in culture (Knutson et_ al_. , 1980a), XB cells, a cloned epithelial line of mouse teratoma cells (Rheinwald et_ al_. , 1975) when co-cultured at high density with irradiated 3T3 mouse fibrablast cells exhibited a keratinization response when exposed to TCDD. Spontaneous keratinization occurs in these cells when they are seeded at low density (.Rheinwald et_ al_. , 1975). High density XB/3T3 cultures not treated with TCDD did not show this response. Other halogenated aromatic hydrocarbon congeners including dibenzo-pdioxins, dibenzofurans, biphenyls and azp(xy)benzenes also induced keratinization in the XB/3T3 system although to a lesser extent than 2,3,7,8 TCDD. Keratinization could also be induced by some non halogenated aromatic hydrocarbons such as benz(a) anthracene and 5,6-benzoflavone (Knutson et_ al_. , 1980). Since 2,3,7,8 TCDD is the most potent, but not the only inducer of this hyperkeratinization response, this effect will be referred to here as "dioxin-like" activity. Unrelated toxins were studied to determine if the keratinization response was due to nonspecific cell damage. The direct acting alkylating agents W-acetoxy-2-acetylaminofluorene and Mi-methyl-N'-nitrosoguanidine; inhibitors of nucleic acid synthesis, actinomycin D, bleomycin and cordycepin; the spindle microtubule inhibitor, colchicine; kepone, a chlorinated hydrocarbon producing a toxic syndrome distinct from that of TCDD; and 2,6-dichlorobenzo-nitrile, and acnegen considered to have a different mechanism �1 * than TCDD all failed to produce keratinization in the XB/3T3 culture system (Knutson et_ aJL , 1980). 3 Furthermore, a correlation has "been demonstrated between the degree of toxicity of various TCDD isomers and congeners and the extent of enzyme induction, receptor landing and hyperkeratinization response (Knutson et al., 1980; Poland et_ al. , 197T; Poland et_ al. , 1976a; Kende et_ al_. , 1971*). The XB/3T3 system is extremely sensitive, giving an induction of keratinization with as little as 3.2 pg of TCDD (Knutson et_al., 1980). In addition, this system is particularly relevant as a model since it deals with an induced differentiation of the intact cell using an endpoint (hyperkeratinization) known to "be relevant to human exposure (i.e., chloracne). This report describes studies which have been carried out to determine the feasibility of using the in vitro XB/3T3 keratinization model as a mass screen of environmental samples for "dioxin-like" activity, i.e., the ability to induce a hyperkeratinization response. Such an assay could be applied to the detection of "dioxin-like" activity in extracts of samples collected at the Binghamton State Office Building. The evaluation and verification of this assay system is being done by the testing of 12 specific soot samples from various floors of the BSOB building and comparing the results with those obtained from high resolution mass spectroscopy of the same samples when they become available. �METHODS Sample Preparation A soxhlet extract of the soot sample in "benzene (Smith et_ al. , 1981) at a volume of 0.5 - 2.0 ml was mixed with 100 yl of dimethylsulfoxide (DMSO) and evaporated at room temperature for 2k- hours in darkness to allow for solvent exchange from benzene to DMSO with a final volume of 100 yl. A solvent exchange was also done with the positive controls (2,3,7,8 TCDD in DMSO) and solvent controls (DM50). The extract, now in DMSO, was diluted 1:1000 in tissue culture medium (Dulbecco's modified Eagles medium supplemented with 20$ fetal calf serum) and four more 10-fold dilutions were made in this medium for application to the cell cultures. This di- lution series was freshly made for each of the twice weekly refeedings. Cells The XB-2 cell line and the 3T3 feeder cells were obtained from H. Green (M.I.T. ) (.Rheinwald et. al_. , 1975). The cultures were grown in Dulbecco's modified Eagles medium with 100 TJ/ml penicillin and 100 yg/ml streptomycin. The XB line was cultured in medium conditioned "by a 2U hour exposure to confluent 3T3 cells (25 ml/ 75 cm2). The medium was supplemented with 20$ fetal calf serum for the XB cells and 10$ for the 3T3 cells. No contamination with mycoplasma was detected by the Hoechst fluorescence staining method (Chen, 197^). Keratinization screen assay for "dioxin-like" activity Initial screening was done essentially as described by Knutson and Poland (.Knutson. _et_ al_. , 1980) for the in vitro keratinization model. Target cells were the XB epithelial clone derived from mouse teratoma cells by Rheinwald and Green (Rheinwald et. al., 1975). When these cells are plated at low cell density (250 cells per 60 mm dish) along with lethally irradiated 3T3 cells (3 X 105 cells per 60 mm dish), the XB cells form epithelial colonies which stain red with Rhodanile blue. Rhodanile blue stains most mammalian tissue blue but keratinized tissues are stained red (MacConaill et al., 196^). When XB cells are plated at high density (lO5 cells/dish) in the pre- �5 sence of irradiated 3T3 cells (3 X 10 cells per dish), the XB cells replicate but 5 keratinization is not seen and therefore the red staining component of Rhodanile blue (Rhodamine B) is absent. Addition of TCDD to cultures of XB cells at high cell density which are refactory to spontaneous keratinization, produces a dose-related keratinization with the associated red staining with Rhodanile blue. TCDD was shown to pro- duce maximal red staining at concentrations of 5 X 10"11 to 1CT7M; 1 X lO'^M TCDD produces an intermediate response while cultures treated with 5 X 10~12M TCDD and untreated control cultures do not keratinize. This response reaches a maximum by day 10-13. When XB/3T3 cells were plated in l6 mm wells containing 1 ml of medium, as little as 3.2 pg of TCDD produced observable keratinization (Knutson et_ aJL., 1980). Confluent cultures of 3T3 cells were lethally irradiated with IfOOO rads by a cesium source and both the irradiated 3T3 and XB cells were seeded together in Costar Multiwell dishes (15 mm) at 5 X 104 XB cells and 5 X 10U irradiated 3T3 cells per ml per well. These dishes were incubated at 37°C in a humidified of 5% COa in air. atmosphere The medium was replaced twice weekly. The dilution series of the sample was added to the assay cultures 2U hr post seeding and with each medium change. Appropriate solvent controls were run as well as 2,3,7,8 TCDD reference samples. After 12 days, the cultures were rinsed with phosplate buffered saline (PBS) fixed for 30 min. in 10% formalin-PBS and stained with 1% Rhodanile blue or 1% Rhodamine B (.Knutson et_ al_, , 1980). The cultures were then observed for the occurrence of red staining material indicative of the keratinization response. The highest dilution of sample capable of inducing keratinization greater than background levels was taken as the endpoint. The endpoint of the 2,3,7,8 TCDD standard was compared to the endpoints derived from the extracts in order to determine the TCDD equivalency of the sample. The range of activity of a sample was calculated from, the endpoint as follows: It has been shown (Knutson et_ al_., 1980) that 10-11M TCDD produces only a moderate keratinization response, (compared to that produced by 10~10M and higher). �6 >•; Furthermore, one half this amount, i.e. 5 X 10~~12M TCDD produces no response at all. Therefore, in all cases production of such a moderate endpoint was taken to indicate the presence of "between 5 X 10~12 and 1 X 10-nM TCDD equivalent. The assumption was made that one fifth the amount of sample which gave the moderate response would give a negative response. Thus the amount of sample which contains "between 5 X 10~12 and 1 X 10~1:1M dioxin-like activity was taken to "be "between 2Q% and 100$ of the actual amount of sample which gave the moderate response. Safety Considerations It is recognized that the safe handling, containment, and disposal of the substances to "be used in this study are essential. Some of the precautions include prohibition of mouth pipetting, subtraction weighting, use of glove box storage cabinets vented with HEPA and charcoal filters, restriction of traffic in hazard-area, no food, smoking or drinking in hazard area, accurate record keeping of hazardous materials from r-eceipt to disposal, use of filter vented biohazard hoods (BioGard Type II B) f°r medium changes and use of commercial hazardous waste disposal facilities. �RESULTS Assay of BSOB Extracts Preliminary studies were performed to attempt to duplicate the in vitro keratinization model in tMs laboratory. XB/3T3 cultures vere exposed to 10~9M TCDD in DMSO or to DMSO alone at 0.01% final concentration to correspond to the level in the TCDD solution. After 12 days of incubation, with refeeding every 3 to h days, these cultures were fixed and stained. Cultures exposed to 10~9M TCDD showed a marked increase in keratin deposition, as shown by intense Ehodamine B staining, as compared to the DMSO negative control [Fig. l). A 10-fold dilution series of authentic 2,3,7,8 TCDD from 10~8 to 10~12M was tested. The minimal concentration capable of inducing a keratinization response greater than background levels was found to be IQ-^M (Fig. 2b). This is equivalent to 3.2pg/ml and is in general agreement with published results for this compound (Knutson et_ al_. , 1980). A BSOB soot extract which had been analyzed by mass spectroscopy for TCDD and TCDF CSmith et_ al., 198la) was also tested in this experiment for its ability to induce a hyperkeratinization response. A dilution series of this extract was applied to the XB/3T3 cell cultures as described in methods. Figure 2a shows that the DMSO solvent control dilution series induced no keratinization. The highest concentration of the DMSO soot extract (l ul/ml, equivalent to 820 ug soot/ml) caused cytotoxicity. The lowest concentration of extract which induced keratinization was 10~3 ul/ml, which is equivalent to 0.82 yg of soot CFig. 2c). From this result the concentration of .dioxin-like activity in the extract vas calculated (as described in Methods)" to be in the range of 2-20 ppm. Analysis of this sample by mass spectrometry (Smith et_al. , 198lb) indicated the presence of 1.2 ppm 2,3,7,8 TCDD and U8 ppm 2,3,7,8 TCDF. 'The latter is 20-fold less potent then 2,3,7,8 TCDD (.Knutson et_ aiU , 1980); therefore, W ppm of this compound is equivalent in keratinization activity to 2.1* ppm of 2,3,7,8 TCDD. Thus, the total keratinization activity,.based on the mass spectrometry data for these two compounds, should be that of 3.6 ppm 2,3,7,8 TCDD. This is within the concentration limits actually ob- tained in the keratinization assay. �• ; 8 In order to further test the ability of the assay to predict the results of mass spectrometric analysis, benzene extracts of 12 soot samples from different floors of the BSOB vere tested for their ability to induce keratinization. Cells were seeded and exposed to dilutions of the extracts in culture medium. The cultures vere refed every 3-^ days with fresh dilutions of the extract. After 21 days, the cultures were fixed and stained with Rhodamine B. It became obvious, however, that a decrease in the magnitude of the hyperkeratinization effect in response to TCBB exposure had occurred, apparently with time, such that induced keratinization could be seen clearly only under microscopic observation. Colonies of keratinizing cells, similar to those described by Rheinwald and Green (1975) could be seen on a background of non-keratinizing cells. (Attempts to determine the reason for this decrease and to regain the earlier level of responsiveness seen in Figure 1 will be described later.) This decrease in magnitude of the keratinization response did not however, decrease the sensitivity of the assay as seen in studies with authentic TCDD. Accordingly, blind microscopic evaluation was carried out on each of three replicates for each sample by two observers for the occurrence of keratinizing colonies. The results, shown in Table 1, are an average of 6 values per sample and showed good reproducibility with variation rarely being greater than one dilution for any of the replicates of any single sample. The solvent controls and blanks showed no false positive response. Calculations for dioxin-like activity were made as described in Methods. The results show a variation of about 3 orders of magnitude in the ^dioxin-like" activity of the various samples. There appears to be something of a trend in that the samples from the lower floors seem in general to have less activity than those from the higher floors. The results of this study will be compared to the mass spectrometric analysis of these samples when this becomes available. The average keratinization results in terms of dioxin-like activity in ppm were plotted against the available PCB analysis data for these samples generated by gas chromatography (Pause, 1981). Figure 3 shows a double log plot of these two sets of data. There appears to be a possible correlation between the PCB content �9 and the keratinization inducing activity of these soot extracts. Possible reasons for this correlation are examined in the discussion section. AttemptG Made to Reverse the Decrease in the Magnitude of the Hyperkeratinization Effect A personal communication from Dr. A. Poland confirmed that a decline in magnitude was also experienced in his laboratory and was alleviated by the use of reconstituted frozen stocks of early passage XB cells. However, such a strategy in this laboratory failed to increase the magnitude of the keratinization endpoint. It has also been shown that the ratio of XB cells to the irradiated 3T3 feeder cells can affect the magnitude of the response (Rheinwald et_ al_. , 1975). Experiments done to optimize this ratio also failed to restore the response to the early levels, as did changing the irradiation time of the feeder cells, changing the refeeding schedule, using culture vessels of different manufacture and lot numbers, changing incubators, altering the pH of the cultures, using stain from various sources or using medium conditioned by unirradiated 3T3 cells rather than co-cultivation of XB cells with irradiated 3T3 cells. Current attempts to increase the magnitude of the keratinization response will be addressed in the discussion section. Morphological Alteration - A Possible Alternative Endpoint The appearance of an altered cell morphology was observed with those cells which had been exposed to TCDD as well as the various soot extracts in the experiment described in table 1. This change was first seen after 7 days of exposure to the samples and was characterized by a flat cell morphology as compared to the more fibroblastoid cells in the unexposed cultures or those exposed to less than 10""11!/! TCDD CFigure U). An apparent cessation in cell growth was also seen in these flat cells, while the unexposed cells continued to proliferate, resulting in more dense cultures. The minimal concentration of each sample capable of inducing the flat cell response was recorded on the 13th day of exposure of the cultures to the extracts. Replicate dilution series were evaluated by phase microscopy of the living cells by two observers generating 10 values for each sample. The agreement between replicates was excellent with variation rarely being greater than one dilution �••; lo for any one sample. One half of the replicates were fixed and stained for a record, while the remaining cultures were later evaluated for the previously described keratinization (table l). The high correlation between the induction of the keratinization response and that of the flat cell morphology'(Figure 5) indicates that it may by possible to use this flat cell morphological change as an alternative and perhaps improved endpoint in the assay for dioxin-like activity. Preliminary experiments suggest that the induction of the flat cell appearence by TCDD is not dependent upon the irradiated 3T3 feeder layer, thus simplifying any proposed assay procedure using this endpoint. The induced flat cell morphology has been found to be apparent after only 6-7 days of exposure to authentic 2,3,7,8 TCDD, thereby allowing the assay to be run without the necessary refeeding needed in the keratinization assay. Effects of Triton X 100 on the Assay Triton 30.00 (TX) has been proposed for use in the decontamination of the BSOB. Since TX is soluble in benzene which is currently used in the soxhlet ex- traction of the samples and could therefore be encountered in samples to be assayed, a preliminary experiment was carried out to deterimine whether TX has any effect on the assay. A concentration range of TX from 10~* - 10~^yl/ml was tested with the standard concentration series of the TCDD standard (10~8 - 10~12M). It was found that TX at 10"1 yl/ml is cytotoxic while levels of 10~2 and 10~3 yl/ml are not. No synergistic or antagonistic effect of TX in regard to the TCDD induced flat cell endpoint was observed. There was also no induction of a false positive result by TX alone at any concentration tested. Experiments to determine the effect of TX on the keratinization endpoint are in progress. �11 DISCUSSION: The fire at the Binghamton State Office Building and involvement of an electrical transformer containing a mixture of PCBs (Araclor 125^-) and chlorinated "benzenes lead to subsequent contamination of the structure with dioxin and dibenzofuran laden soot. A need was established for a rapid semiquantitative assay for "dioxin-like" activity. This would allow prioritization of samples for high resolution quantitative and qualitative analysis in order to monitor the progress, of decontamination. This preliminary report has dealt with the evaluation and verification of such an assay which is based on an in vitro model of the in vivo keratinization response to dioxin exposure. Preliminary studies demonstrated that the published in vitro model was reproducible in this laboratory and that this model was sensitive to levels of dioxin (2,3,758-TCDD) which corresponds to that in the literature (Rnutson et al, 1980) (approximately 10 M). Assay results of a single soot extract for which mass spectrometry data were available indicated a good correlation between these data and the data resulting from the keratinization mo'del. This initial verification lead to the application of this assay to 12 soot extracts for comparison with results of mass spectrometry of the same samples as they become available. However, the decrease in the magnitude of the TCDD induced keratinization effect may reduce the usefulness of this particular endpoint in spite of the readily apparent, microscopically observable, induction of keratinizing colonies. Attempts are currently being made to redone the XB cell line for the desired characteristics of hyperkeratinization upon TCDD exposure/ Successful monitoring of the cell line, cell storage and recloning would insure a stable cell population upon which the keratinization assay could be based. Cloning is being done by identification and isolation of spontaneously keratinizing colonies of XB cells in sparse culture, as well as keratinizing colonies of XB cells induced by TCDD �12 exposure in dense cultures. New XB and 3T3 feeder cell starter cultures will be obtained, since the reconstitution and use of our early passage frozen stocks has failed to increase the magnitude of the keratinization response to that seen in the initial assays. The subjective reading of the endpoint by visual estimation of the minimal concentration of extract capable of keratin induction based on the intensity of Rhodamine B staining or identification of keratinizing cell colonies limits the degree of quantitation possible. Therefore, several other methods of achieving better quantitation of the induced keratin production are being considered and evaluated. One involves extraction of the Rhodamine B stain from the cells and quantitation by fluorometric measurement. Another involves a quantitative radio- immunoassay method for mammalian epidermal keratin protein which has recently been published (Yuspa et_ al, I960). Another possible method for detection of increased keratin production to be explored is based upon the observation (Steinert et al_, 19T8) that in keratinizing cells, keratin represents 20% of the total urea extractable cellular protein. synthesized. Thus, keratin may represent the predominent protein being Furthermore, keratin has been shown to be relatively rich in histidine, methionine and cysteine (Belanger, 1956; Clark, 1968; Hambrick, 1966). Thus, it may be possible to quantitate keratin production by measurement of incorporation of these (radiolabeled) amino acids into cellular protein. The verification of the flat cell morphological change as an indicator of "dioxin-like" activity would allow a second and perhaps improved biological endpoint to be used in a routine screen assay in addition to or in place of the keratinization assay. The Endpoint is apparent after only 6-7 days and can be readily assessed by microscopic observation of living cultures'. Another advantage of the short development time of this endpoint is the lack of the necessity to refeed the cultures, thus reducing both the sample requirement and handling of the cultures. The homo- geneity of these cells may allow use of smaller culture vessels, reducing even further �13 the amount of sample needed. In addition recent experiments have suggested i that the irradiated 3T3 cell feeder layer may not "be needed for the development of this endpoint. Further studies vill be carried out to establish whether the induction of the flat cell morphology is a valid endpoint for the detection of dioxin-like activity. These will include a double blind study to test the ability of the endpoint to quantitate various concentrations of authentic 2,3,798-TCDD and a test of the relative activities of other halogenated polycyclic compounds, e.g. TCDF, biphenylene, PCB, azo(xy)benzenes, as well as nonhalogenated aromatic hydrocarbons. Compounds that inhibit cell proliferation by acting on macromolecular synthesis vill also be tested to determine if the flat cell response in these cells is simply due to the coincidental inhibition of cell growth. Attempts to establish cloned cell lines exhibiting the flat cell characteristic to insure homogeneity are currently underway. The possible correlation which we have observed between the in vitro assay results and the PCB content of the soot samples (Figure 3) lead to concern that the PCBs were responsible for the induction of keratinization. In fact, it has been reported that 2,3,^,2' ,3' ,^' polybrominated biphenyl can induce keratinization in the XB/3T3 system at a concentration 2,000 fold higher than 2,3,7,8TCDD (Knutson et al, I960). To examine this possibility, Araclor 125U, the PCB mixture present in the transformer coolant and thus the presumed source of PCBs in the soot samples, was tested for .the induction of the flat cell morphology. The lowest concentration of Araclor 125^ for which the flat cell induction was observed vas 10 yg/ml. The highest concentration of PCBs found in any of the soot samples tested was 23,000 yg/gm (Pause, 1981}> The lowest amount of this sample which gave an endpoint was 1.5 yg of soot per ml which contains 0.035 Pg of PCBs per ml. Based upon the results with Araclor 125U, this is insufficient to induce the flat cell response. Thus, the observed induction of the flat cell morphology by the soot extracts is unlikely to have been caused by the PCBs contained in the �lit samples. Experiments are in progress to determine if similar results are obtained for PCB induced keratinization. Another explanation for the correspondence "between the PCB data and the in vitro assay results may be a synergistic or additive interaction between PCBs and low levels of TCDD or some other substance in the soot samples. Studies designed to detect such interaction between Araclor 125^ and 2,3,7,8-TCDD are being carried out and studies using PCBs to spike actual soot extracts are planned. The correspondence could also be the result of a similar deposition pattern of the PCBs and the dioxin congeners and isomers in the building. Triton X-100, the proposed decontamination agent for the BSOB, was found to be toxic to the in vitro system at 0.10 yl/ml but not at 0.01 yl/ml. TX is soluble in benzene, and a relatively low, non toxic, concentration in a sample could be concentrated by a benzene extraction procedure to toxic levels and preclude use of the in vitro assay. This possibility should be considered in the development of decontamination and sample collection procedures. The lack, of effect of TX on the induction of the flat cell response does however, decrease the concern of a false positive result from low levels of this compound. In summary, the in vitro keratinization model developed by Knutson and Poland (Knutson et al, 1980) has been examined for use as a screen assay for the detection of "dioxin-like" activity in extracts of BSOB soot samples. After initial verification with authentic TCDD, a preliminary assay was done on the one sample for which TCDD and TCDF data from mass spectrometry'were available. The results of this in vitro assay compared well with the quantitative analysis data. Based on this result, a screen for keratinization induction was carried out on 12 extracts of soot from various floors in the BSOB. These results will be compared to the mass spectrometry data on the same samples, thus allowing a more rigorous validation. A high correlation would indicate that this model may be �-.; 15 used as a semiquantitative mass screen assay for "dioxin-like" activity. The decrease in the magnitude of the keratinization response which we have observed with time gives reason for concern, as the cause for this decline has not been determined. The problem may be solved by the acquisition of new starter cultures, or stabilized for a time by recloning the line for the desired characteristics and/or using reconstituted frozen stocks of those cultures which currently exhibit TCDD induced keratinizing colonies. Alternatively, the TCDD- • induced change in morphology (which is characterized by the appearance of flat, non proliferating cells) which seems to correlate very well with the keratinization endpoint may have the potential for use as an additional or alternative (and possibly better) endpoint for use in mass screening for "dioxin-like" activity. �16 REFERENCES Belanger, L.F. (1956) Autoradiographic Visualization of the Entry and Transit of 6^5 Methionine and Cystine in the Soft and Hard Tissues of the Growing Rat. Anat. Rec_., 12^:555-563 Bradlaw, J.A., and Casterline, J.L. (1979) Induction of Enzyme Activity in Cell Culture: A Rapid Screen for Detection of Planar Polychlorinated Organic Compounds. J_. Asspc. Off. Anal. Chem., 62:90^-915 Chen, T.R. (197*0 Utilization of Fluorescent Hoechst Stain to Effectively Detect Mycoplasma Contamination. In Vitro 10:390 Clark, S.L. (1968) Incorporation of Sulfate by the Mouse Thymus: Its Relation to Secretion "by Medullary Epithelial Cells and to Thymic Lymphopoiesis. J_. Exp. Med. , 128, 927-957Fukuyama, K. and Epstein, W.L. (1966) Epidermal Keratinization: Localization of Isotopically Labeled Amino Acids. J_. Inves. Derm., kj, 551-559. Hambrick, G.W., Lamberg, S.I. and Bloomberg, R. (1966) Observations on Keratinization of Human Skin In Vitro. £. Inves.Derm., Vr, 5^1-550. Hay, A. (i960) Health monitoring endangered at Sev&so. Nature, 283, 237. Hutzinger, 0., Olie, K., Lustenhouver, J.W.A., Okey, A.B., Bandiera, S., and Safe, S. (1981) Polychlorinated Dibenzo-P-Diozins and Dibenzofurans: A Bioanalytical Approach. Chemosphere 10, 19-25. Kende, A.S., Wade, J.J., Ridge, D. and Poland, A. (197*0 Synthesis and Fourier Transform Carbon-13 Nuclear Magnetic Resonance Spectroscopy of New Polyhalodibenzo-p-dioxins. J_. Org. Chem., 39, 931-937. Kimbrough, R.D., Coleman, D.C., Liddle, J.A., Cline, R.E. (1977) Epidemiology and Pathology of a Tetrachlorodibenzodioxin Poisoning Episode. Arch. Environ. Health, March-April, 77-85. Knutson, J.C., and Poland, A. (1980) Keratinization of Mouse Teratoma Cell Line XB Produced by 2,3,7,8-Tetrachlorodibenzo-p-dioxin: an in vitro model of toxicity. Cell, 22, 27-36. Knutson, J.C. and Poland, A. ( 9 0 2,3,7,8-Tetrachlorodibenzo-p-dioxin: 18) Failure to Demonstrate Toxicity in Twenty-three Cultured Cell Types. Toxicol. and Applied Pharmacol., 5^, 377-383. Langer, H.G., Brady, T.P. and Briggs, P.R. (1973) Formation of Dibenzodioxins and other Condensation Products from Chlorinated Phenols and Derivatives. Environ. Health Perspect., 5, 3-7. Lowery, O.K., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) Protein Measurement with the Folin Phenol Reagent. J_. Biol_. Chem., 193, 265-275. �17 MacConaill, M.A. , and Gurr, E. (19610 The Histological Properties of Rhodanile Blue. Ir_. J_. Med. Sci . , June, 2^3-250. Pause, R. ( 1981 ) Personal Communication, N.Y. State Department of Health. Pocchiari, F., Silano, V. and Zampieri , A. (1979) Human Health Effects from Accidental Release of TCDD at Seveso, Italy. Ann. N.Y. Ac ad. Sci. 320, 311-320. Poland, A. and Kende, A. (1976) 2,3,7»8-Tetrachlorodibenzo-p-dioxin: environmental contaminant and molecular probe. Fed. Proc., 35 , Poland, A., Glover, E., Kende, A.S., DeCamp, M. , and Giandomenico, C.M. (I976a) 3,^,3' jV-tetrachloroazoxybenzene and azobenzene: Potent Inducers of Aryl Hydrocarbon Hydroxylase. Science , 19^, 627-630. Poland, A. and Glover, E, (1977) Chlorinated Biphenyl Induction of Aryl Hydrocarbon Hydroxylase Activity: A Study of the Structure -Activity Relationship. (1977) Mol. Pharmacol., 13, 921+-938. Reggiani, G. (1978) Medical Problems Raised by the TCDD Contamination in Seveso, Italy. Arch. Toxicol. , UO, l6l-l88. Rheinwald, J.G. and Green, H. (1975) Formation of a Keratinizing Epithelium in Culture by a Cloned Cell Line Derived from a Teratoma. Cell* 6, 317-330. Smith, R.M., O'Keefe, P.¥., Hilker, D.L., Jelus-Tyror, B.L., and Aldous, K. ( 9 l Chemical analysis of 2,3,7,8-tetrachlorodibenzo-p-dioxin and 2,3,7,8I8) tetrachlorodibenzofuran in a soot sample from the transformer explosion in Binghamton, New York. N.Y. State Department of Health Report. February 20, 198l. Smith, R.M., Hilker, D.L., O'Keefe, P.W., Kumar, S., O'Brien, J., Jelus-Tyror, B.L., and Aldous, K. (I98la) Analysis of a Binghamton soot sample for tetrachlorodibenzofurans and tetrachlorodibenzo-p-dioxins . N.Y. State Department of Health Report, October 1, 1981. Steinert, P. and Yuspa, S.H. (1978) Biochemical Evidence for Keratinization by Mouse Epidermal Cells in Culture. Science , 200, Yuspa, S.H., Poirier, M.C., Harness, J.R., Olsom, D.R. and Steinert, P.M. (1980) Specific Quantification of Mouse and Human Keratin Proteins by Radioimmunoassay. Bio_chem. J_. , 187 » 281-28U. �18 Table I Keratinization Assay of BSOB Soot Samples Floor number Lowest active c o n c e n r a o n owes a c v e concentration of extract (pg soot equivalent/ma)a 3 k 6 1.3 llt.O 15.9 7 8 0.3 1.0 9 10 12 Ik 15 17 1.5 0.5 0.7 12.9 O.It. O.k TCDD , v equivalent ,(ppm) 0.01 1.2 0.11 0.10 - 0.10 - 12 - 1.1 - 1.0 5.3 - 53 1.6 - 16 l.l 3.2 2.3 0.12 k.O k.O - 11 - 32 - 23 - 1.2 - kO - kO a) Activity was determined by the appearance of keratinizing colonies. This result is the average of values from 3 replicates evaluated by two observers. b) .Calculated as described in Methods. �Figure Legends Figure 1 Effect of 2,3,7,8 TCDD on XB/3T3 Cell Cultures Cultures were seeded and cultivated as described in methods. Figure la is representative of an XB/3T3 culture exposed to 10r* M 2,3,7,8 TCDD in the presence of 0.01$ DMSO. Figure Ib is representative of a replicate culture exposed to 0.01$ DMSO alone. The bright red rhodamine B stain, indicative of keratin deposition} shows as dark grey in these photos. Figure^ 2 Keratinization assay of a BSOB soot extract Figure 2a shows the negative response of the DMSO control. Figure 2b shows the effect of 2,3,Tj8 TCDD on this system with keratinization first evident with 10"1:LM. Figure 2c shows the toxic effect of a BSOB soot extract at the highest concentration (l yl/ml) and the induction of keratinization at lower concentrations, with the effect first detected at the fourth highest dilution (10~3 ul/ml), equivalent to 0.82 yg soot/ml. Figure 3 Log-log plot of the amount of PCBs detected in BSOB soot extracts from various floors by gas chromatography (abscissa) and the calculated values of "dioxin-like" activity generated by the keratinization assay of the same samples (ordinate). The values for "dioxin-like" activity were derived by dividing the amount of soot per ml in the lowest soot extract concentration capable of inducing keratinization into 3.2 pg/ml which is the lowest concentration of 2,3,7>8 TCDD found to induce keratinization. Figure k TCDD induced flat cell morphology Figure Ua: XB/3T3 culture after 12 days of incubation with 0.1$ DMSO, showing high density and fibroblast-like organization. Figure li-b: a similar culture exposed to 10~°M TCDD in 0.1$ DMSO, showing flat morphology and relative low density. Phase, l^OOX magnif ifcation. Figure 5 Log-log plot of calculated values of "dioxin-like" activity of BSOB soot extracts from various floors generated by the keratinization assay (ordinate) and by the induction of the flat cell response (abscissa) calculated as in Figure 3. �20 Figure la Figure Ib �21 X .* • •! - o •-- - o' = • Figure 2a Figure 2b �22 Figure 2c �23 4-J •H n •u o «j rt O cd -H OT ?*! a8 p< to ,3 I 1 2 i 3 log ppm PCB in soot samples Figure 3 t 4 JE �Figure 4a Figure 4b �.3 4J 0 I 4J 3 ctf »H W I <U 0 log ppm "dloxin-like" flat cell induction in soot samples Figure 5 � Dublin Core The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/. Title A name given to the resource Alvin L. Young Collection on Agent Orange Description An account of the resource <p style="margin-top: -1em; line-height: 1.2em;">The Alvin L. Young Collection on Agent Orange comprises 120 linear feet and spans the late 1800s to 2005; however, the bulk of the coverage is from the 1960s to the 1980s and there are many undated items. The collection was donated to Special Collections of the National Agricultural Library in 1985 by Dr. Alvin L. Young (1942- ). Dr. Young developed the collection as he conducted extensive research on the military defoliant Agent Orange. The collection is in good condition and includes letters, memoranda, books, reports, press releases, journal and newspaper clippings, field logs and notebooks, newsletters, maps, booklets and pamphlets, photographs, memorabilia, and audiotapes of an interview with Dr. Young.</p> <p>For more about this collection, <a href="/exhibits/speccoll/exhibits/show/alvin-l--young-collection-on-a">view the Agent Orange Exhibit.</a></p> Text A resource consisting primarily of words for reading. Examples include books, letters, dissertations, poems, newspapers, articles, archives of mailing lists. Note that facsimiles or images of texts are still of the genre Text. Box The box containing the original item. 088 Folder The folder containing the original item. 2212 Series The series number of the original item. Series IV Subseries II Dublin Core The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/. Creator An entity primarily responsible for making the resource Gierthy, John F. Gerald D. Frenkel Title A name given to the resource Typescript: A Preliminary Report on the Evaluation of an In Vitro Assay for the Detection of "Dioxin-Like" Activity Using Extracts of Soot from the Binghamton State Office Building, 1982 Subject The topic of the resource BSOB PCBs analytical studies https://www.nal.usda.gov/exhibits/speccoll/files/original/626cc9118cfe8eb74b4354189c0cbb55.pdf a7600869caa44e1fd59a725e2d453e51 PDF Text Text Item D Number °2208 Author Gierthy, John F. Corporate Author Report/Article TltlO Progress Report: Evaluation of an In Vitro Assay for the Detection of "Dioxin-Like" Activity in the Binghamton State Office Building Journal/Book Title Year Month/Day Color Number of Images October D 7 DescrlptonNotoa Thursday, September 20, 2001 Page 2208 of 2293 �Progress Report: Evaluation of an In Vitro Assay for the Detection of "Dioxin-Like" Activity in the Binghamton State Office Building by John F. Gierthy and Gerald D. Frenkel New York State Department of Health Center for Laboratories and Research October, 1982 RECEIVED OCT 1 3 1932 DIRECTOR PUBLIC HEALTH �Abstract In an earlier report the application of the cell keratinization assay (CKA) to BSOB soot extracts as a test for polychlorinated dioxins, dibenzofurans and biphenylenes was described. The ability of the test to rank order the extracts with regard to level of contamination suggested that the CKA may be used as a screen for dioxin-like activity. A problem arose however, concerning a decline in the ability of the cells to respond to TCDD. This problem has now been alleviated by technical modificatons of the original protocol. Further development and verification of what appeared to be a TCDD-induced change in cell morphology ("flat cell" induction) as an alternative assay has also been carried out. Experimental results indicate that this assay shows the same sensitivity to 2,3,7,8 TCDD and 2,3,7,8 TCDF as the CKA. Furthermore, this morphological change appears to be specific for the particular epithelial cell line used in this procedure and is not induced by various other toxic compounds including inhibitors of DNA, RNA and protein synthesis, and mitosis. These results indicate the specificity of flat-cell induction for dioxin isomers and congeners. Twenty nine benzene extracts of cotton gauze BSOB swipes, prepared by Versar, have been tested using the CKA and flat-cell assays. Results using an internal standard of 2,3,7,8 TCDD indicate loss of activity during the solvent exchange. This may be related to the appearance of a precipitate during the solvent exchange from benzene to DMSO. �-1Introduction In the preliminary report of January, 1982 the development and verification of an assay for dioxin-like activity was described (1). The cell keratinization assay (CKA), is based on the ability of 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD) to induce keratinization in a specific line of epithelial cells grown in culture as originally described by Knutson and Poland (2). Extracts (3) of soot samples taken from ceiling panels of various floors of the BSOB were tested for dioxin-like activity in the CKA and the results were compared to the actual polychlorinated dibenzodioxin (PCDD) and polychlorinated dibenzofuran (PCDF) content of the samples as determined by high resolution GC/MS chemical analysis. The results of this comparison are not contained in the report of January, 1982 because the chemical analysis had not yet been completed. They were, however, presented at the meeting of the BSOB Expert Panel on March 29, 1982 in Binghamton (4). Briefly the level of dioxin-like activity, as determined in the CKA correlated well with that predicted by the chemical analysis. The results of the CKA permitted ranking of the samples with regard to dioxin-like activity, which corresponded accurately to the levels of PCDDs and PCDPs in the samples. These results suggested the potential use of the CKA to screen a large number of samples and rank them with regard to their degree of contamination with PCDDs and PCDPs. A problem associated with the CKA also became evident, however, during the course of these experiments. The cells exhibited a progres- sive decline in the magnitude of their keratinization response to 2,3,7,8 TCDD exposure. This decline called into question the utility �-2- of the CKA in an ongoing screening program. Described below is the progress which has been made in solving this problem. The preliminary report also contained a description of a morphological change which the cells underwent, apparently as a result of exposure to PCDDs and PCDFs. It was suggested that this change might form the basis of an alternative assay ("flat cell assay") for dioxin-like activity. However, very few compounds had been tested using this end- point; thus, although the results appeared to correlate well with results obtained from the CKA, the specificity of the alternative assay was very uncertain. Described below is progress which has been made with regard to this question. Results a) Modification of CKA New starter cultures of the XB epithelial and 3T3 feeder layer cells were obtained from Dr. H. Green (5). These cultures were propo- gated and portions were frozen for later use. Previously, cells which had been frozen and thawed were not responsive in the CKA (1). The freezing procedure was modified from that used previously (1), resulting in the recoverability of responsive cells. It is thus now possi- ble to maintain a large supply of frozen cells which can be thawed and propogated when needed. It was also found that an increase in the ratio of 3T3 to XB cells to 10 resulted in an enhanced keratinization response. It now appears that with these two technical modifications, the CKA can be used on an ongoing basis. �-3- b) Validation of the Flat-cell Assay Since the endpoint for this assay is determined by a subjective determination of altered cell morphology, the reproducibility of the assay as well as its ability to quantitate concentrations of 2,3,7,8 TCDD in various samples was established in a double blind experiment. The results indicated that the reproducability and sensitivity of this assay is comparable to that of the CKA. Various inhibitors of macro- molecular synthesis and mitosis were examined for their ability to induce the flat cell morphological change. Hydroxyurea (a DNA synthesis inhibitor), actinomycin D (a RNA synthesis inhibitor), cycloheximide (a protein synthesis inhibitor) and colchicine (a mitosis inhibitor) were all found to be inactive over a broad range of concentrations. This indicates that the flat cell effect is not associated with a general toxicity but may be a specific effect, analagous to, and perhaps related to, keratinization. This possibility is supported by the finding that 2,3,7,8 tetrachlorodibenzofuran (TCDF) also induced the flat cell effect, but had approximately one tenth the potency as 2,3,7,8 TCDD, which is the relative potency which has been reported for these two compounds in the induction of keratinization (2). Fur- thermore, other cell lines, including fibroblasts and transformed epithelial cells, were tested for their ability to respond to TCDD with a change in morphology, and no flat cell effect was observed. Thus, the specificity of the flat-cell assay appears, from these preliminary experiments, similar to that of the CKA in terms of both inducing compounds and susceptible cells. �-4- c) Application to BSOB samples Twenty nine benzene extracts of cotton gauze swipes of various locations in the BSOB were received from Versar. These were processed in a manner analogous to the soot samples which had been analyzed previously (1): one ml of a benzene extract was solvent exchanged to 50 ul of DMSO and a dilution series applied to the cells. However, it was noted that after the solvent exchange, a precipitate appeared in the sample extracts and matrix blank extracts but not in the reagent blanks. The precipitate was removed (with some difficulty) by centrifugation. All samples, including those which had been spiked with authentic 2,3,7,8 TCDD gave either very low or no activity in the CKA and the flat-cell assay. Experiments are currently being done to determine the cause of this loss of activity. Among the possibilities which are being tested are that a) there is a loss of activity during the solvent exchange procedure, and b) material in the cotton gauze is extracted by the benzene, but is insoluble in DMSO and the active dioxins and furans adsorb to the precipitated material. �-5- References 1. Gierthy, J.F. and Frenkel, G.D. (1982). A preliminary report on the evaluation of an In Vitro assay for the detection of "dioxinlike" activity using extracts of soot from the Binghamton State Office Building. New York State Deparment of Health Report, January, 1982. 2. Knutson, J.C. and Poland, A. (1980). Keratinization of mouse cell line XB produced by 2,3,7,8-tetrachlorodibenzodioxin: an in vitro model of toxicity. 3. Cell, 22, 27-36. Smith, R.M., Bilker, D., O'Keefe, P.W., Kumar, S., Aldous, K., and Jelus-Tyror, B. (1982). Determination of polychlorinated dibenzofurans and polychlorinated dibenzodioxins in soot samples from a contaminated office building. New York State Department of Health Report, March, 1982. 4. G. Eadon, K. Aldous, G. Frenkel, J. Gierthy, D. Bilker, L. Kaminsky, P. O'Keefe, J. Silkworth and R. Smith (1982). Compari- sons of chemical and biological data on soot samples from the Binghamton State Office Building. March, 1982. 5. Rheinwald, J.G. and Green, H. (1975). Formation of a keratinizing epithelium in culture by a cloned cell line derived from a Teratoma. Cell, 6, 317-33 � Dublin Core The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/. Title A name given to the resource Alvin L. Young Collection on Agent Orange Description An account of the resource <p style="margin-top: -1em; line-height: 1.2em;">The Alvin L. Young Collection on Agent Orange comprises 120 linear feet and spans the late 1800s to 2005; however, the bulk of the coverage is from the 1960s to the 1980s and there are many undated items. The collection was donated to Special Collections of the National Agricultural Library in 1985 by Dr. Alvin L. Young (1942- ). Dr. Young developed the collection as he conducted extensive research on the military defoliant Agent Orange. The collection is in good condition and includes letters, memoranda, books, reports, press releases, journal and newspaper clippings, field logs and notebooks, newsletters, maps, booklets and pamphlets, photographs, memorabilia, and audiotapes of an interview with Dr. Young.</p> <p>For more about this collection, <a href="/exhibits/speccoll/exhibits/show/alvin-l--young-collection-on-a">view the Agent Orange Exhibit.</a></p> Text A resource consisting primarily of words for reading. Examples include books, letters, dissertations, poems, newspapers, articles, archives of mailing lists. Note that facsimiles or images of texts are still of the genre Text. Box The box containing the original item. 088 Folder The folder containing the original item. 2208 Series The series number of the original item. Series IV Subseries II Dublin Core The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/. Creator An entity primarily responsible for making the resource Gierthy, John F. Gerald D. Frenkel Date A point or period of time associated with an event in the lifecycle of the resource 1982-10-01 Title A name given to the resource Progress Report: Evaluation of an In Vitro Assay for the Detection of "Dioxin-Like" Activity in the Binghamton State Office Building Subject The topic of the resource BSOB dioxin PCDD