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Ehctm"mental Health Perspectives Vol. 80, pp. 101-108, 1989 PLAINTIFF'S EXHIBIT DOW-1543 Mechanisms of Asbestos-Induced >quamous Metaplasia in Tracheobronchial Epithelial Cells y Gregory Cameron,* Craig D. Woodworth,* >usan Edmondson,* and Brooke T. Mossman* ST0272762 Within 1 to 4 weeks after exposure to asbestos, differentiated rodent and human tracheobronchial epithelial cells in organ culture undergo squamous metaplasia, a putative preneoplastic lesion characterized by con version of mucociliary cell types to keratinizing cells The exogenous addition of retinal acetate (RA) to cul ture medium of hamster tracheal organ cultures reverses preestablished, asbestos-induced squamous metapla sia. although data suggest that the effectiveness of RA decreases as the length of time between exposure to asbestos and initial application of RA increases. o-Difluoromethylomithine (DFMO), an irreversible inhibitor of omithlne decarboxylase (ODO, inhibits squamous metaplasia caused by asbestos or vitamin A deficiency, whereas addition of methylgiyoxal bistguanylhydrazone) (MGBG), a structural analog of spermidine and inhibitor of S-adenosylmethionine decarboxy lase, causes an enhancement of metaplasia under both circumstances Basal cell hyperplasia and increased incorporation of 'H-thymidine by tracheal epithelial cells also are seen after addition of the polyamines, putrescine or spermidine, to tracheal organ cultures, an observation supporting the importance of polyamines in the development of this lesion. The use of retinoids and inhibitors of ODC could be promising as preventive and/or therapeutic approaches for individuals at high risk for development of asbestos-associated diseases. Introduction "Asbestos" refers to a family of hydrated silicates of fi brous (> 3:1 length:diameter ratio) dimensions. Occupa tional exposure to these minerals has been linked to the development of pulmonary fibrosis (asbestosis), mesothelioma, and lung cancer (i.e., bronchogenic carci noma) (;). The latter disease is of critical importance as it has an extremely poor prognosis and is the cancer type associated with the highest mortality rate in man. Both epidemiologic and experimental data suggest that asbestos is a cocarcinogen and/or tumor promoter in the development of bronchogenic carcinoma (2). For example, in comparison to smokers in the general population (8- to 10-fold increased risk of bronchogenic carcinoma), non smoking asbestos workers have a 1.5- to 4-fold increased risk of lung cancer. In contrast, asbestos workers who Department of Ethology, University of Vermont College of Medicine, Burlington, VT 05405 Reprint requests should be addressed to Brooke T. Mossman, Dept, of Pathology, University of Vermont College of Medicine, Soule Alumni Bldg., Burlington, VT 05405. smoke have a more striking risk (80- to 92-fold) of disease (5). With the exception of the rat, most animals do not de velop bronchogenic carcinoma after inhalation or in tratracheal instillation of asbestos unless polycyclic aro matic hydrocarbons (PAH), chemical carcinogens in cigarette smoke, are adsorbed to the surfaces of the fibers U). Thus, asbestos appears to act as a cocarcinogen by delivering PAH to tracheobronchial epithelial cells (5), the progenitor cells of bronchogenic carcinoma. Tumor promotion by asbestos has been demonstrated in rat tracheal grafts exposed previously to noncarcinogenic amounts of the PAH, dimethylbenzanthracene (6). Subsequent insertion of asbestos into these grafts causes the development of tumors, whereas neoplasms are not observed after application of identical concentrations of DMBA or asbestos alone, lb elucidate possible mechan isms of asbestos-induced tumor promotion in the respira tory tract, work in this laboratory has focused on the bio logic effects of asbestos on hamster tracheal epithelial cells in monolayer and organ culture. Many of the changes reported in cultured cells exposed to phorbol esters, clas sical tumor promoters studied extensively in mouse skin, are observed in tracheal epithelium after addition of as- Noncfi mis maiebai mat e moiecteo r COPYRIGHT LAW (TITLE 17 U. S. CODE). 10J CAMERON ETAL. C 9 1 Z IZ U S- bestos These include stimulation of plasma membrane Reversion of Squamous Metaplasia i . : ' ,, marker enzymes (7), increased cell division (8,9), increased activity of ODC (9), and development of hyperplastic and metaplastic changes (8,10-12). Understanding the pathogenesis of squamous metapla sia is of particular relevance to the development of bron chogenic carcinoma, as the lesion is considered an inter mediate step in the progression of morphologic events leading to neoplasia. Although squamous metaplasia as sociated with trauma or vitamin A deficiency is revers ible, it is unclear whether metaplastic changes caused by chemical or physical carcinogens, such as asbestos, resolve with time or develop into malignancies For example, squamous metaplasia is observed commonly in the respiratory tract of smokers, a group at high risk of developing bronchogenic carcinoma (13). The results of studies from this laboratory suggest that asbestos-induced squamous metaplasia occurs when fibers impinge on the tracheal epithelium (8,10-12). Fibers then cause sloughing of superficial cells and com pensatory regeneration of epithelial cells that are squa- Tracheal organ cultures were divided equally into two groups with three treatment regimens (A, B, and C) as indicated in Figure l. Each group contained untreated tracheas (A); explants exposed to benzo(a]pyrene (BaP) (0.5 pg/mL medium, dissolved in acetone at a final concen tration of 0.1% in medium) three times per week for 3 weeks (B); and tissues exposed to crocidolite asbestos (UICC reference sample)(4 pg/mL medium) for 1 hr at the time of initiation of cultures (C) (6,8,9,11). Each of the treatment groups was further subdivided with the sub divisions receiving no retinal acetate (RA), or RA (Sigma Chemical Company) (dissolved in dimethyl sulfoxide at a final concentration of 0.1% in medium) at 10*7 M or 10"' M three times per week for 1 week. Groups I and 2 received the RA at different times. Whereas group 1 received the RA for 1 week at week 3 of culturing (To), group 2 received the RA for 1 week at - 5 weeks (Ti). All explants were harvested at the end of - the RA treatments. 1 l - mous in nature. Whereas smaller fibers are phagocytized f successfully by macrophages and epithelial cells, longer Prevention of Squamous Metaplasia O, fibers appear to act as matrices for proliferation of cells over their surfaces Thus asbestos-induced metaplastic le sions occur at localized sites of accumulation of fibers on the epithelial surface, unlike the broad expanses of metaplasia observed in vitamin A deficiency (lk). Work here was initiated to determine if retinoids, i.e., synthetic derivatives of vitamin A, could reverse preestablished squamous metaplasia in hamster tracheal or gan cultures exposed to crocidolite asbestos or the PAH, benzo(a)pyrene (BaP). Because retinoids appear to in fluence polyamine and DNA synthesis (15), we also inves tigated the ability of various inhibitors of polyamine bi osynthesis to modify squamous metaplasia caused by vitamin A deficiency or exposure to asbestos Last, the polyamines putrescine, spermine, and spermidine were added to tracheal organ cultures to determine if they caused increased DNA synthesis as measured by incor poration of JH-thymidine, in tracheal epithelium. Hamster tracheal organ cultures were prepared as described and divided into groups (n = 9-15 ex plants/group). Tb determine whether inhibitors of poly amine biosynthesis affected squamous metaplasia caused by asbestos (protocol #1), untreated controls and explants exposed initially for 1 hr to crocidolite asbestos (4 mg/mL medium) were maintained in MEM with and without ad dition of DFMO (5 mM, Merrell National Labs) or MGBG (5 pM, Merrell National Labs). Medium with and without drugs was replenished three times weekly. An additional group received DFMO (5 mM) followed at 24 hr by MGBG (5 pM) three times weekly. In other experiments, nonasbestos-exposed cultures were maintained in Waymouth's MAB 87/3 medium (GIBCO) with the addition of insulin (1 pg/mL medium), hydrocortisone (0.1 pg/mL medium), and antibiotics This formulation results in a complex vitamin A-deficient medium causing squamous metaplasia (16). DFMO (1 or Materials and Methods 5 mM), MGBG (5 pm), or DFMO (1 pm) followed by MGBG (5 pm) at 24 hr was added to designated cultures three Preparation of Tracheal Organ Cultures times weekly (protocol #2). The technique for preparation and culture hamster tracheal explants has been described in detail previously (16). In brief, female golden Syrian hamsters (6-8 weeks of age) were sacrificed by IP injection of sodium pentobar bital, and the tracheas dissected and cleaned of surround ing tissue. After the tracheas were opened longitudinally, they were cut again in half and sectioned into double ring explants. Tissues were divided into groups and cultured in 35-mm plastic culture dishes containing 4 to 5 explants per dish. The explants were maintained in 0.5 mL serumfree Minimum Essential Medium (MEM) (GIBCO) sup plemented with 100 pg/mL gentamycin and 25 units/mL nystatin (16). Cultures were incubated at 37C in an at mosphere of 95% air and 5% CO2 and the culture medium changed three times per week. Autoradiographic Studies We reported previously (17) an increase in the extent of squamous metaplasia in hamster tracheal organ cul tures exposed to putrescine (1 mM) in culture medium, lb determine if addition of polyamines would increase basal cell hyperplasia as measured by incorporation of JHthymidine in tracheal epithelium, tracheal organ cultures were prepared as described above and maintained in Waymouth's MAB/873 with additives The medium in selected groups then was supplemented with putrescine, spermi dine, or spermine (all at 1 and 10 mM; Sigma Chemical Company) three times weekly for 3 weeks At this time, organ cultures (n = 14-19/group) were pulsed for 5 hr with JH-thymidine (10 pCi/mL medium) (New England ASBESTOS-INDUCED SQUAMOUS METAPLASIA o 1 WHS Exptjnts Established Retmyl Acetate Treatment Croups: A) Untreated B) BAP (O.S^g/ml 3X/wk X 3 wk*) c) UICC Crocidolite (IX, a mg/ml X 1 hr) * WKS T Harvest IOJ ST0272764 5 WKS 6 WKS Treatment Croups: Retinyl Acetate (1X/1 wk. Id'7 M. Id'* M) A) Untreated 8) BAP (O.S^g/ml 3X/wk X 3 wks) C> UICC Crocidolite (tX. it mg/ml X t hr) fe 1. Protocols for evaluation of whether retina) acetate reverses squamous metaplasia in control (A), BaP exposed (B), and asbestosposed (C) hamster tracheal organ cultures. dear i before preparation for histology as described be. Ur.itair.ed 5 sections were prepared for autoradi, aph> as described previously (8) and assessed by light .roscopy for numbers of epithelial cells incorporating i-thymidine. istology and Grading of Squamous ietaplasia All organ cultures were fixed in 10% buffered formalin, nbedded in paraffin, sectioned at 5 pm, and stained with cmatoxylin and eosin. The grading system for scoring le extent of metaplasia has been reported previously (8). briefly, the prepared sections were examined by light microscopy and given a score depending on the extent of metaplasia observed. Cultures showing normal differen tiation and no metaplasia were given a score of 1. Ex plants with focal metaplastic lesions that covered less than 15% of the epithelial surface were considered as a 2. If the metaplasia covered more than 15% but less than 50% of the epithelium, the explant was scored as a 3. Ex plants with metaplasia covering more than 50% of the epithelium were scored as 4s. Slides were coded and scored independently by two in vestigators, and the scores were averaged. In studies us ing RA to inhibit squamous metaplasia, data were ana lyzed by a multiway analysis of variance with the metaplastic score treated as the dependent variable and the other factors (dosage, time, and treatment) adjusted for in the analysis (/S). The Kruskal-Wallis analysis was used to evaluate metaplastic changes in organ cultures ex posed to polyamines and inhibitors of polyamine biosyn thesis (18). Results and Discussion Effects of RA on Squamous Metaplasia A number of studies have demonstrated the importance of vitamin A in maintaining the normal differentiation of tracheobronchial epithelium (li,19-22). In the absence of vitamin A, the mucociliary' epithelium converts to squa mous metaplasia, a lesion also observed after trauma (23,24) or exposure to toxic agents (1 2,25). After addition of chemical carcinogens, squamous metaplasia occurs in organ cultures of many types of epithelial cells (26-28). Both prevention and reversal of these lesions have been achieved after addition of retinoids to culture medium (lit,29-32). Retinoids also appear effective in preventing the development and growth of chemically induced tumors in laboratory animals (33-36) although their mech anising) of action is unclear. Asbestos is a physical carcinogen causing squamous metaplasia in the tracheobronchial epithelium (8,10-12) and is associated with an increased risk of bronchogenic carcinoma in man (/). Although chronic administration of the retinoid retinyl methyl ether prevents the appearance of asbestos-induced metaplasia in hamster tracheal organ cultures (<S), the question of whether squamous metapla sia can be reversed after establishment of the lesion has received little attention. Accordingly, we addressed the questions: a) Can retinoids reverse preestablished, asbestos-associated squamous metaplasia? b) Does the time interval between the addition of asbestos and appli cation of a retinoid affect the potential of the retinoid to reverse squamous metaplasia? and c) Can retinoids re verse squamous metaplasia induced by a chemical carcino gen such as BaP in the tracheal bioassay? Tb determine the effects of retinoids on various treat ment groups over extended time periods, it is of critical importance that time in culture has no effect on the de velopment of metaplasia. Figure 2A, which is compiled from the pooled data of the 3 treatment groups (control, BaP, and asbestos), shows that length of time in culture from 4 to 6 weeks does not influence the extent of metaplasia. In contrast to the situation observed with as bestos, BaP does not cause a significant increase in squa mous metaplasia in hamster tracheal organ cultures (Fig- 10. CAMEROS' ET AL ure ZB). This observation supports our previous work (37) in which an increase in squamous metaplasia was found in cultures exposed to various concentrations of BaP over a 4-week period. Although metaplastic lesions were ob served sporadically in the presence of BaP, these changes were not reproducible When data from all test groups is pooled, administra tion of RA results in a dosage-dependent decrease in the amount of squamous metaplasia at all time periods (Fig ure 3A). Additionally, all groups respond similarly to the retinoid, although the absolute amount of squamous metaplasia is more stiking, regardless of the concentra tion of retinoid, in the asbestos group as compared to con trol and BaP exposed explants (Figure 3B). A 3 I 0) < S' < * 0 0 to** RA (cone) 10" ST0272765 A 3 UOxJ Z 2 to < < UJ 2 B A o a O A ASBESTOS BAP O CONTROL A 8 0 J------ ,------------------------------- --r46 TlME(weci s) B CONTROL BAP treatment FtCl'RE 2. Effect of time in culture LAl and exposure to 8aP or asbestos (fl) on the development of squamous metaplasia in trachea] organ cul tures. Time in culture (A) had no effect on the amount of metaplasia observed in the 3 test groups Treatment of explants with asbestos (8) significantly increased the amount of metaplasia observed (p < 0.001). whereas exposure to BaP djd not cause an increase in metapla sia in comparison to control explanta The dose of RA and treatment group 04) and dose of RA and time (8) were adjusted for in the statisti cal analyses. o__ ,,, o 10'* I0'f RA (cor*c) Figure 3. Effectiveness of RA m reversing squamous metaplasia in tracheaJ organ cultures. Under all circumstances, RA reversed squa mous metaplasia in a dose-dependent manner (p < 0.001) {A), al though the relative amount of squamous metaplasia was more strik ing m asbestos-exposed explants (0). Statistical analyses were adjusted for time and treatment in (A), whereas time alone was ad justed for in (B). Figure 4 suggests that the effectiveness of RA decreases as the length of time between exposure to as bestos and initial application of RA increases. This obser vation is supported by the results of in vivo studies of others showing that delayed administration of retinoids leads to their diminished effectiveness in preventing mammary tumor growth (38,38). Unlike chemical carcino gens that are metabolized by tracheal epithelial cells, as bestos fibers are insoluble and remain entrapped in metaplastic lesions for 6 weeks and longer in culture. Thus, asbestos-induced squamous metaplasia appears to be more persistent and is reversed less effectively by reti noids than lesions associated with exposure to soluble tox icants. Effects of Inhibitors of Polyamine Synthesis on Squamous Metaplasia 0DC is the first and rate limiting enzyme in the biosyn thesis of polyamines (Figure 5), essential growth regula- ST0272766 ASBESTOS INDUCED SQUAMOUS METAPLASIA 105 3 sA 8w(0 2 8 UJ A asbestos 3 BAP O CONTROL 0 ____,-------------------------------- ,---------46 TMEI.otks) ,1'RE 4. Effect of time on the efficacy of RA in reversing squamous metaplasia. Data suggest that RA is less effective in reversing asbestos-induced metaplasia when administered at S weeks in com parison to 3 weeks (p < 0.09). ry molecules. The ability of phorbol compounds to injce ODC is related directly to their potency as tumor lomoters, thus the induction of the enzyme is thought i be essential to the process of tumor promotion (40). In jpport of this hypothesis, DFMO, a specific, noncompeti,ve inhibitor of ODC, inhibits promotion in a number of Kpenmental models including mouse skin (41), colon (42), ancreas (43), and mammary gland (43). Whereas DFMO nduces differentiation of various tumor cell lines (4 7,48), t inhibits differentiation of preadipocytes (49) and myo)lasts (50). RA also inhibits tumor promotion in mouse ,kin (45), ODC induction, levels of polyamines, and proliferation in mouse skin (15) and cultured cells (46). Tb determine whether inhibitors of polyamine synthe sis could inhibit squamous metaplasia in vitamin Adeficient trachea! organ cultures or explants exposed to croeidolite asbestos, we added DFMO, MGBG, a struc tural analog of spermidine and an inhibitor of 5- adenosylmethionine decarboxylase (51) (Fig. 5), or DFMO followed by MGBG to culture media three times weekly. Under the latter circumstances, DFMO increases the up take of MGBG by cells (52). As shown in Figure 6, exposure to croeidolite asbestos or MGBG alone results in increased amounts of squamous metaplasia (p < 0.05) in comparison to controls. A higher percentage of explants exhibiting extensive squamous metaplasia is observed in the group exposed to crocidolite asbestos and MGBG, an observation supporting a pos sible additive effect of agents. In contrast, an increase in metaplasia was not observed in groups exposed to croeidolite with addition of DFMO, DFMO alone, or DFMO in combination with MGBG. Thus, DFMO appears to inhibit both asbestos- and MGBG-induced squamous metaplasia. Data provided in Figure 7 show no effects of DFMO on the development of metaplasia in vitamin Adeficient tracheal organ cultures, whereas MGBG alone or MGBG in combination with DFMO augment squamous metaplasia significantly (p < 0.05). Thble 1 illustrates the effects of various polyamines on DNA synthesis when added to the medium of tracheal or gan cultures over a 3-week period. The addition of putrescine (1 and 10 mM) and spermidine (1 mM) cause signifi cant (p < 0.05) increases in numbers of epithelial cells incorporating 'H-thymidine, whereas spermine (1 mM) does not enhance the normal labeling index. Both sper mine and spermidine were cytotoxic, as determined by histopathology, to tracheal epithelium at 10 mM (data not shown). The data suggest that the polyamines, putrescine and spermidine, enhance epithelisd cell replication and the de velopment of squamous metaplasia in hamster tracheal organ cultures Although increased proliferation of many eukaryotic cells has been observed after addition of putrescine and spermidine to monolayer cultures (53,54), COIOH ORNITHINE H,N(CH,)jCHNH, DFMO Ornithine decarboxylase -CO, L-METHIONINE 4- ATP 4MGBG. - S-AOE NOS YL METHIONINE S- Adenos ylmerhion*nc dccarboxy'ase CO, PUTRESCINE H,N(CH,).NH, Spermidine synihase SPERMIDINE H,N(CH,),NH(CH,1 ,NH, S-METHYtAOENOSYl- homocysteamine Seer mme synthase SPERMINE H,NtCH,),HN(CH,).NH(CH,),NH, ME thylthio ADENOSINE Ficure 5. Diagram illustrating biosynthesis of polyamines. toe CAMCROX ST AU CO L < w. (0 0 O^mMgObSgMU*** Cro0c*MO0H<M MG0G \% m Q Figure 6. Effects of DFMO and MGBG on squamous metaplasia in duced by asbestos Addition of crocidohte (4 mg/mL) or MGBG (5 pM) caused an increase in squamous metaplasia in comparison to controls whereas no increase was observed with addition of crocidolite in com bination with DFMO (5 mM). Asterisk (*) denotes p < 0.05. Table 1. Incorporation of 'H-thymidine in Hamster tracheal epithelium after addition of polyamine*. Groups % Labeled n epithelial cells* Control Putrescine, 10 mM Putrescine. 1 mM Spermine. I mM Spermidine. 1 mM 19 14 19 16 15 2.42 <- 0.64 7.28 1.80" 6.96 1.31" 2.36 - 0.69 6.95 - 0.68" 'Mean SE. 100 epithelial cells from each of five serial sections were counted for each explant. increased in comparison to untreated controls (p < 0.05). the finding that these polyamines augment basal cell hyperpiasia and metaplastic differentiation of trachea) epithelial explants is novel. The inhibition of asbestosinduced 3quamous metaplasia by DFMO, a drug deplet ing de novo synthesis of all polyamines (55), further strengthens the hypothesis that polyamines are critically involved in the induction of squamous metaplasia by as bestos. For reasons that are unclear, DFMO did not ap pear to inhibit the squamous metaplasia observed with vitamin A deficiency or vitamin A deficiency in combina tion with MGBG (Fig. 7). In comparison to putrescine and spermidine, spermine appears to be relatively less impor tant in cellular proliferation as growth inhibition only oc curs when intracellular pools decline to < 60% of normal (55). However, this polyamine appears to play an impor tant physiological role in intracellular calcium homeosta sis (55). Although MGBG can block synthesis of both sper mine and spermidine, it also increases cellular transport of extracellular polyamines, even in the presence of DFMO (51). Thus, increased accumulation of putrescine and/or spermidine under these circumstances might ex- FIGURE 7. Effects of DFMO and MGBG on squamous metaplasi^jjij tracheal organ cultures maintained in a complex medium encourag ing squamous metaplasia. The addition of MGBG (5 txM) or DFMO ,. mM) in combination with MGBG caused a significant increase in squamous metaplasia in comparison to control cultures; asterisk (*) denotes p < 005 plain the enhanced amount of squamous metaplasia observed in MGBG-treated explants. In conclusion, depletion of polyamines by DFMO or treatment with retinoids appear to be an effective means of preventing and/or reversing asbestos-associated squa mous metaplasia in vitro. The use of these agents may be rewarding as prophylactic or therapeutic approaches to lung disease in man. tI \ \ 1I Wbrk from this laboratory is supported by grant ROl CA33501 from NCI. Many individuals have contributed to these studies including Michael Bergeron. Judith Kessler. 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