Carcinogenesis by Nonmutagenic Chemicals: Early Response of Rat Liver Cells Induced by Methapyrilene1

(CANCER RESEARCH 45, 2184-2191, May 1985]

P. Thomas type,2 CorazÃ³n D. Bucana,3 and Susan P. Kelley

LBI-Basic Research Program, Chemical Carcinogenesis Program, NCI-Frederick Cancer Research Facility, Frederick, Maryland 21701

ABSTRACT mammalian cells (2, 8), and does not induce unscheduled DNA synthesis (6, 25, 30) or sister chromatid exchanges (20) in a Although methapyrilene (MP) produces hepatocellular carci number of mammalian cells. It has also been reported by Lijinsky nomas in rats, it does not elicit many of the cellular responses and Muschik (22) that tritiated MP does not bind to genomic induced by other hepatocarcinogens. We have investigated the DNA of rat liver to any significant extent. However, Althaus ef al. early changes induced in rat liver epithelial cell cultures by MP (1) have shown that DNA damage can be induced by this using morphological, cytochemical, and cytofluorometric tech carcinogen in primary cultures of rat liver cells. Therefore, it niques. Within 2 h of MP treatment, inclusion bodies which were appears that, although MP may not directly affect nuclear DNA, stainable with lipid stains were observed in the cytoplasm. Ultra- it may still produce at least minor alterations in the genomic structurally, they resembled lamellar bodies with alternating light makeup and/or on gene expression of target cells. and dark lamellae. These bodies were transient in nature, since In this study, we report the immediate effects of MP and other they disappeared within 24 h of removal of MP. They were, related compounds on their target cells. Similar experiments, however, retained in the cytoplasm as long as MP was present using well-characterized, diploid epithelial cells from rat liver, in the meduim. Lamellar bodies appear to be induced in the were also done with other hepatocarcinogens and drugs chem presence of histamine H1 receptor-blocking agents, since meth- ically related to MP or which produce similar biological responses aphenilene and diphenhydramine produced this reaction, but in animals. It was found that MP and other histamine H1 receptor- cimetidine, an H2 antagonist, did not. blocking agents induced an accumulation of lipid particles which Morphologically, mitochondria of control cells were long and were retained in the cytoplasm until the drug was removed. rod-like, whereas they were short or bizarrely shaped in the MP- Moreover, a distinct increase in the total mitochondrial content treated cells. Moreover, a quantitative increase was observed in and in the percentage of binucleated cells was noted in the MP- the mitochondrial content of the treated cells, when intact liver treated liver cell cultures. cells vitally stained with Rhodamine 123 were analyzed with a fluorescence-activated cell sorter. A significant increase in binu- MATERIALS AND METHODS cleated cells was observed when liver cells were exposed for 10 to 12 days with MP. Collectively, these results suggest that MP Test Chemicals. MP, clofibrate, DMBA, and AFL were purchased might perturb the cytoskeletal elements leading to an alteration from Sigma Chemical Co. (St. Louis, MO). Diphenhydramine hydrochlo in the nuclear and mitochondrial makeup of rat liver cells. ride (Benadryl) was a product of the Parke-Davis Co. (Ann Arbor, Ml). Nitrosodiethanolamine, methaphenilene, and cimetidine were supplied by Dr. W. Lijinsky (Frederick Cancer Research Facility, Frederick, MD), and INTRODUCTION Compound Wy 14643 was obtained from Dr. A. Palmer (Frederick Cancer Research Facility). Rhodamine 123 was purchased from Eastman Kodak It is now well established that many 'nongenotoxic" chemicals Co. (Rochester, NY). induce tumors in different species and tissues (11, 12, 18, 21, Cell Cultures. Two well-characterized liver epithelial cell cultures were 24). One such agent, MP," has been shown to be a potent used in these studies. One (FNRL-1), derived from a normal 12-day-old hepatocarcinogen in rats after long-term feeding (13, 24, 28, 31). Fischer rat, and the other (FPHL-1), derived from an 18-month-old Some of the early responses induced by MP in rat liver cells in partially hepatectomized Fischer rat, were established and kept frozen in this laboratory and used between passages 10 and 20 in these studies. vivo have already been reported. These include an increase in The establishment, lineage, and characteristics of these liver cell cultures the number of mitochondria and other morphological changes in have been described in detail previously (17). Both are diploid epithelial periportal cells (28, 31 -33), some of which may be a reflection cells and expressed none of the characteristics of transformed liver cells of toxicity. MP is generally considered to be a nongenotoxic during the period of this study. In addition, a malignant liver cell line, chemical, since it does not produce mutations in bacteria or in derived from FNRL-1 by AFL treatment in vitro (19), and primary liver cells, isolated by the methods described (17) from rats gavaged with test 1Research sponsored by the National Cancer Institute, Department of Health and Human Services, under Contract N01-CO-23909 with Utton Bionetics, Inc. chemicals (see below), were also used in some of the experiments. Culture Methods. Stock cultures were maintained in Ham's F-12 The 1.2 million-V electron microscope at the NIH High Voltage Electron Microscopy Biotechnology Resource, Center for Laboratories and Research, New York State medium (14), supplemented with 10% fetal bovine serum (Sterile Sys Department of Health, Albany, NY, was used in this study. tems, Inc., Logan, UT), and were subcultured at weekly intervals using 2To whom requests for reprints should be addressed. 3 Present address: Department of Cell Biology, M. D. Anderson Hospital and 0.05% trypsin (206 units/mg protein; Millipore Corp., Freehold, NJ). Cells were plated in 60-mm plastic culture dishes (Falcon Plastics, Oxnard, Tumor Institute, Texas Medical Center, 6725 Bertner Avenue, HMB 173, Houston, TX 77030. CA) at a seeding density of 4 x 103/sq cm, which produced a confluent 4The abbreviations used are: MP, methapyrilene hydrochloride; AFL, aflatoxin monolayer in 7 days. Bi: DMBA, 7,12-dimethylbenzanthracene; Wy 14643, 4-chloro-6-oxylidino-2-pyr- Experiments in Vitro. FNRL-1 and/or FPHL-1 cells were plated either emidithioacetic acid; TEM, transmission electron microscopy or micrograph; HVEM, high-voltage electron microscopy. at very low seeding density (20 cells/sq cm), so that they would produce Received 6/25/84; revised 11/19/84; accepted 1/24/85. distinct cell colonies in 10 days, or at the regular seeding density (4 x

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cm), to produce a confluent monolayer in 7 days. Cells were area of MP-treated samples compared with the control (Fig. 1, a treated with MP in concentrations from 0 to 100 Â¿tg/ml(MP stock versus b). Such granules were seen 24 h after treatment with solutions in distilled water were diluted with the culture medium) either MP, within a dose range of 10 to 100 iig/ml. Even treatment 1 day after plating or when the cells were nearly confluent. Cells were with doses as low as 2.5 Â¿Â¿g/mlproducedgranulation, but only examined and photographed using a Zeiss inverted-phase-contrast mi after a longer period of incubation (48 h). A dose of 100 nQÂ¡m\ croscope at different times after treatment. Similar experiments were has been shown to be toxic to the liver epithelial cells because also done using AFL, DMBA, nitrosodiethanolamine, clofibrate, Wy it inhibited the colony-forming ability of these cells (20). In all 14643, methaphenilene, diphenhydramine, and cimetidine. Cell colonies formed from the sparse cultures were projected on a further experiments in this study, we used a concentration of 25 television screen through a video camera fixed on a Ziess microscope /Â¿g/ml,a dose which produced considerable granulation within with a x 10 objective. Four colonies selected at random from each quarter 24 h without any appreciable effects on the viability of the cells, of the control and MP-treated dishes (4 dishes each) were used for as studied by colony-forming ability and/or acute morphological counting the total as well as binucleated cells. changes. These granules were stainable with the lipid stains, Cells grown on Formvar-coated coverslips and fixed with 3% glutar Sudan Black B and Oil Red 0. When fixed cells were examined aldehyde plus 2% p-formaldehyde in 0.1 M cacodylate buffer (pH 7.35) with Nomarski optics, the untreated control cells showed distinct were also examined with Nomarski interference optics. In addition, nucleus, nucleolus, and rod-shaped mitochondria in the perinu samples were also fixed at various times (2 to 24 h) and processed for electron microscopy. Cells which had been pretreated with MP for 24 h clear area (Fig. 1c). In contrast, cells that had been treated with and then allowed to "recover" for various periods in the regular culture MP for 24 h showed numerous granular structures in the peri- medium without MP were also processed for TEM. nuclear area of the cytoplasm, and rod-shaped mitochondria In some experiments, the control and MP-treated cells were vitally were seldom seen. stained with the mitochondrial-specific stain Rhodamine 123 (9). The Further experiments on the induction of lamellar bodies by MP cells were incubated for 15 min with 6 fig of Rhodamine per ml in the were done using only phase-contrast microscopy. Cytoplasmic culture medium and then postincubated for 15 min in the medium without granulation could be induced by MP in both proliferating (sparse the dye. The cells were examined and photographed using a Zeiss culture) and in nonproliferating (confluent monolayer) liver cell Universal microscope with epifluorescence attachment with an excitation- cultures. When sparse cultures of FNRL-1 cells were treated barrier filter and a reflector combination (excitation, 400 to 490 nm; barrier filter, 520 nm; reflector, 510 nm). In addition, Rhodamine-stained continuously with MP for 12 days, the cytoplasmic lipid granules persisted throughout the period, but they still disappeared within cells were trypsinized, and the cell suspensions were analyzed using a fluorescence-activated cell sorter (FACS IV; Becton and Dickenson, 1 to 2 days after MP removal. To find out whether this response Sunnyvale, CA) with an argon laser in the 488-nm band. A total of 1 x is a specific characteristic seen only in the 2 diploid liver epithelial 106 cells from both the control and MP-treated cell populations was used cells in culture, we have done similar qualitative in vitro experi for the analyses. ments using other cell cultures (primary cultures of rat liver cells, Experiments in Vivo. Fischer F-344/CR rats (6-month-old males; secondary cultures of rat embryo fibroblasts, a malignant liver average weight, 375 g) from the Animal Resources Program of the cell line derived from the FNRL-1 cells following AFL treatment), Frederick Cancer Research Facility were used. Rats were treated with but no electron microscopy was performed. All these cells did either MP (dissolved in water; 100 ^g/g body weight) or water by gavage produce cytoplasmic granules when examined by phase-contrast (3 times at 9-h intervals) and used 3 h after the final gavage for the isolation of hepatocytes. The livers were perfused with collagenase- microscopy, and the granules disappeared a day after the cells were fed with the regular culture medium. Moreover, freshly dispase as described in detail before (17). Hepatocytes from both the isolated liver cells from a rat gavaged with MP (see "Materials control and MP-treated rats were examined under a phase-contrast and Methods" for details) also showed lipid granules in the microscope, soon after the isolation and again at 24 h after maintaining them as primary culture. cytoplasm, which was not seen in cells isolated from untreated Ultrastructural Studies. Liver epithelial cells plated on a plastic surface control rats. As in the in vitro experiments, these granules or directly on Formvar-coated gold grids were treated with MP (25 ng/ induced by MP in vivo also disappeared when the cells were ml). Samples were fixed with a fixative containing 3% glutaraldehyde maintained for 1 day in vitro without added MP. A number of plus 2% p-formaldehyde in 0.1 M cacodylate buffer (pH 7.35) for 1 h or chemicals were then tested (within a dose range of 10 to 100 with 1% tannic acid in 1% glutaraldehyde for 20 min at room temperature. fig/ml) to see whether they would induce lipid accumulation. The samples were postfixed with 1% osmium tetroxide for 1 h, rinsed 3 These drugs included hypolipidimic agents (clofibrate, Wy times with buffer and distilled water, stained en bloc with 1% aqueous 14643), "genotoxic" carcinogens (AFL, DMBA), another nongen- uranyl acetate for 45 min, and then processed (35). Thin sections were stained with Reynold's lead citrate for 3 min and examined in a Hitachi otoxic carcinogen (nitrosodiethanolamine), and other antihista- HU-12A at an accelerating voltage of 75 kV. minics (methaphenilene, diphenhydramine, cimetidine). Of these, Cells grown directly on Formvar-coated gold grids were fixed using only methaphenilene and diphenhydramine produced lipid accu the osmium thiocarbohydrazide osmium procedure and critical point dried mulation in the liver cell cultures with a dose >5 M9/ml. (5). The gold grids with whole mounts were then examined in an Hitachi We then investigated the effect of MP on the diploid rat liver HU-12A at an accelerating voltage of 100 kV and in an Hitachi H-800 at epithelial cells in greater detail using TEM. In thin sections from an accelerating voltage of 200 kV in both the transmission and STEM the control cultures, hardly any inclusion bodies were seen, but modes. Some of the whole-mount and thick sections (0.25 to 0.5 nm they did contain a number of rod-like mitochondria (Fig. 1e). In thick) were also examined using the high-voltage electron microscope at the MP-treated cells, there was a definite increase in the cyto the NIH Biotechnology Resource Facility at Albany, NY. plasmic granules which resembled lamellar bodies with alternat ing light and dark lamellae (Figs. W and 26). Apparently, there RESULTS was also an increase in the number of mitochondria in the MP- Phase-contrast microscopy of living epithelial liver cells re treated cells (Fig. 1/), but they were smaller and were ovoid, in vealed a large number of cytoplasmic granules in the perinuclear contasi to the rod-like mitochondria seen in the control. From

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Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1985 American Association for Cancer Research. CELLULAR RESPONSES INDUCED BY METHAPYRILENE these sections, it was not possible to determine whether the 2000 small ovoid mitochondria represented tangential sections of rod- like mitochondria which had been placed in a different spatial configuration as a result of the treatment with MP. Therefore, we prepared whole mounts of intact cells (and also thin sections 1500 where needed) and analyzed them using HVEM. Using these techniques, we then investigated the time course of the appear ance and disappearance of lamellar bodies as well as the rela tionship between these cytoplasmic inclusions and mitochondria. Lamellar bodies could be seen in TEM of whole mounts of 1000 cells as early as 2 h after treatment with MP (Fig. 2, d versus c), and their number increased considerably throughout the incu bation. After 6 h in the presence of MP, small multiple bodies enclosed in double membranes were seen (Fig. 2a). An abun 1 500 dance of lamellar bodies was observed both in the whole mounts (Fig. 2e) and in thin sections (Fig. 2b) of liver epithelial cells treated for 24 h with MP. However, 24 h after the removal of the chemical, hardly any lamellar bodies were visible (Fig. 2f). Even as soon as 6 h after removal of MP, the number of lamellar 100.0 150.0 200.0 250.0 bodies was reduced considerably (Fig. 3a).

MP also produced appreciable changes in the morphology and 1000 number of mitochondria. Whole mounts of normal liver epithelial cells in culture exhibited long rod-like mitochondria when exam ined by HVEM (Fig. 2c). Pleomorphic mitochondria were ob served within 2 h after MP treatment (Fig. 2, d versus c), and some bizarre forms were seen in some of the cells treated for 500 24 h (Fig. 2e). Many more smaller ovoid mitochondria were also present in the 24-h sample (whole mounts), compared with the untreated control (Fig. 2, e versus c). Therefore, some of the small ovoid mitochondrial profiles seen in the thin sections of cells treated with MP for 24 hr (Figs. 1r"and 2b) might represent 50.0 100.0 150.0 200.0 250.0 cut edges of the bizarre (branched or hairpin-shaped) mitochon Channel Number Chart 1. Profilesfrom fluorescence-activatedcell sorter of scatter (a) and fluo dria present in the intact cells (Fig. 2e). There does seem to be rescence (b) derived from FNRL-1 cells vitally stained with Rhodamine 123. a real increase in the number of ovoid mitochondria in the treated and , control and MP-treated cells, respectively. An increase in channel cells. Within 24 h after removal of MP, the morphology of almost number represents a larger cell size for a and an increasein mitochondrialcontent all the mitochondria had reverted to the rod-like form (Fig. 2f) forÃ². seen in the control cells (Fig. 2c). However, 6 h after MP treatment, both the small, as well as the rod-like, forms of Table 1 mitochondria were visible (Fig. 3a). To determine whether there Induction of binucleation in rat liver cells by methapyrilene(25 ^g/ml) is any connection between the mitochondria and the lamellar of binu cells/colonyDish31Total no. of cleated cells/col cellsControl1.8of binucleated bodies, stereo photographs of high-voltage (1000 kV) electron onyControl19 micrographs were prepared. When viewed stereoscopically, these 2 organelles were found to be located in different planes treated954 treated48 treated5.2 (Fig. 3b). Â±136 + 4 Â±9 Â±0.8 + 1.0 We then stained mitochondria in the intact, living liver cells 2 1905 Â±219 2043 Â±490 14Â±3 63 Â±6 0.8 Â±0.2 3.7 + 0.8 60 + 14 with Rhodamine 123, a vital stain for these subcellular particles 3 1750 Â±437 1410 Â±492 27 Â±8 1.5 Â±0.2 5.0 + 1.3 4Control1417Â±284b866 Â±196MP 797 + 109No.24 Â±4MP41 Â±6% 1.8 Â±0.8MP5.7 + 1.6 (8), to determine whether there was any increase in the actual mass of mitochondria in the MP-treated cells and not just in the Avc 1484 Â±168 1300 + 203 21 Â±3 53 Â±5d 1.8 Â±0.3 4.9 Â±0.6rf a Four colonies were selected at random from each quarter of the Petri dishes. number of small mitochondria. When examined with a fluores Dishes 1 and 2 were from one experiment, and Dishes 3 and 4 were from another. cence microscope, cells treated with MP for 24 h appeared to 6 Mean Â±SE. contain a greater amount of mitochondria (Fig. 3, c versus d). 0Average of pooled data (16 colonies from each group). Similarly, an increase in the Rhodamine 123-stained mitochondria " Nestedanalysisof variancehas shown a highly significant(P> 0.002)induction of binucleated cells (per colony and percentage of total) by MP treatment. There was observed in the primary cultures of hepatocytes, derived were no significant differences between the values obtained within or among the from a rat that was gavaged with MP. When the control and MP- plates. treated FNRL-1 cell cultures were analyzed with the fluores cence-activated cell sorter, it was found that, while the cell size content was statistically significant (p > 0.005) if the cells were remained similar in both cell populations (Chart 1a), there was a treated for 48 h with MP (25 M9/ml). definite increase in the number of cells with a higher level of Another observation on the liver epithelial cell cultures, FNRL1 fluorescence in the MP-treated cell population (Chart 10). This and FPHL1, was the induction of binucleated cells by MP (Table increase in the number of cells with a higher mitochondrial 1). While MP did not affect the colony size (total number of cells/

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Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1985 American Association for Cancer Research. CELLULAR RESPONSES INDUCED BY METHAPYRILENE colony), a significant (<0.002) induction was observed in the tubules were disrupted with Colcemid (15). It is possible that MP number of binucleated cells/colony or in the percentage of binu- could have brought about the changes in the mitochondria! cleated cells within any of the colonies. Nested analysis of morphology by preferentially interacting with the microtubules variance has also shown that there are no significant differences and/or intermediate filaments or by acting directly on the mito between the data obtained from within or among the experi chondria. The latter possibility is consistent with previous reports ments. Even in other experiments done with a higher inoculum that autoradiographic grains seen in experiments using [3H]MP size of the cells, unit areas of the MP-treated dishes showed a were located in the cytoplasm (20) and were associated with higher percentage of binucleated cells (data not shown). Another mitochondria (32). It is now becoming increasingly clear that a qualitative observation was the frequent occurrence of tripolar number of carcinogenic chemicals bind preferentially with mito- mitoses in the MP-treated cells, while such mitoses were hardly chondrial DNA (3, 27, 40, 41), and the extent of this binding can ever seen in the control liver epithelial cell cultures. be even 40 to 90 times greater compared with the binding to nuclear DNA. Therefore, it is conceivable that MP could produce DISCUSSION significant alterations in the mitochondrial DNA, which in turn would alter the mitochondrial functions, as has been shown with A number of nongenotoxic chemicals have been identified, other hepatocarcinogens (4,10). and others are being added to the list (7,10,11,18,21, 23, 25). Although mitochondria have been implicated in the formation Although other cell systems were used to study the possible of lamellar bodies (34), our TEM studies (Fig. 2a) as well as the mechanisms by which MP might induce cancer, we concentrated stereophotographs from HVEM (Fig. 3b) have shown no indica our efforts to determine the effects of MP on 2 diploid rat liver tions of any association between these 2 inclusions or the direct epithelial cell cultures at early passage levels, before they exhibit formation of lamellar bodies from mitochondria. any transformed phenotypes. These cultures have been shown As MP treatment continued, the mitochondrial number (Fig. (19) to induce the expression of conventional transformed phe notypes after treatment for 8 weeks with the genotoxic hepato- 3o) and their mass increased (as reflected in the increased carcinogen AFL. In the present study, which spanned only 2 to fluorescence with Rhodamine 123, which stained the mitochon 12 days, 3 types of cellular responses to MP were seen: these dria specifically; Chart 10). Previous morphological studies done include the appearance of phase-dense cytoplasmic granules on the liver of rats fed MP have shown a definite increase in the number of hepatic mitochondria (28, 31-33). In a sequential (lamellar bodies) in the perinuclear area; alterations in the mor phology and content of mitochondria; and an increase in the study, Ohshima et al. (28) described an increase in mitochondria number of binucleated cells. in the MP-treated hepatocytes, but most of the adenomas and The appearance of lamellar bodies soon after MP treatment premalignant foci showed a decrease in mitochondrial content. indicates that this may be due to a direct perturbance of normal In contrast to a decrease in mitochondrial content observed after lipid metabolism rather than as a result of genetic expression. feeding rats with a number of potent hepatic carcinogens (29), a However, hypolipidemic agents (clofibrate, Wy 14643) or other definite increase was observed earlier in the liver of rats fed the carcinogenic chemicals (AFL, DMBA, nitrosodiethanolamine) did marginally active aminoazo dye, 2-methyl-4-dimethylaminoazo- not induce this change. This cellular response appears to be benzene (29, 39). specific only for histamine H1 receptor antagonists (MP, meta- Although it has not been proven, the induction of a large phenilene, diphenhydramine), since it was not induced by cimet- number of binucleated cells by MP seems to suggest a pertur idine, an H2 receptor blocker. This response was also transient bation in the microtubules. Recently, a number of nonmutagenic in nature and was not linked to the proliferative status of the carcinogens or tumor promoters have been shown to induce cells, since the cytoplasmic granules could be produced in both aneuploidy (12, 36, 37). Our observations, that binucleated cells dividing and nondividing liver cell cultures. are induced by MP and that mitochondria which are attached to In previous ultrastructural studies in rats, the presence of the fibrillar structure are disrupted by MP treatment, are reason lamellar bodies in liver has not been described. However, in one able indicators that MP may indeed produce aneuploidy after study (31), extensive cytoplasmic vacuolization has been found continuous treatment. in the liver of rats fed 1000 ppm of MP in food. In our experiments, The evidence that MP does not affect nuclear DNA has come we administered divided doses of MP by gavage, so as to avoid primarily from a number of short-term in vitro assays (6, 8, 20, too little or too much MP in the body at any given time. The 25, 30), each with its own limitation and/or differing sensitivity. freshly isolated hepatocytes did contain lipid granules, and par None of these assays could identify a chemical that indirectly affin sections of liver tissue fixed with 4% formaldehyde did show affects DNA of the target cells. It has been reported recently that cytoplasmic vacuoles (data not shown), which does suggest that nonmutagenic tumor promoters produce sister chromatid ex lipid accumulation has occurred. Mitochondrial alterations were also seen within 2 h of MP changes in target cells, when added along with human phago treatment (Fig. 2d), and the morphological changes may have cytes in vitro (38). The differences in published results from been caused by a direct action on the mitochondria or on the assays using hepatocyte primary cultures (1, 6, 8, 25, 30) may fine fibrillar network in the cytoplasm. It is known that mitochon be explained if the different preparations of the freshly isolated dria are arranged along the cytoplasmic microtubules in mono- cells contained liver macrophages (Kupffer cells) and/or leuko layer cultures of various normal cell lines (15); it has also been cytes. Such cells could be activated by MP in vitro (through suggested that a subpopulation of intermediate filaments can histamine H1 receptors; 16). Further research into this aspect of serve as anchorage sites for mitochondria (26). Mitochondria the problem is needed for elucidation of the mechanism of action have been shown to be redistributed in the cytoplasm, if micro- of nonmutagenic carcinogens.

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Fig. 1. a and b, phase-contrast micrographs of live cultures of liver epithelial cells, FNRL-1. a, control untreated cells; b, MP-treated cells. Note the presence of numerous phase-dense granules in the perinuclear area of cells treated with 25 jig of MP per ml for 24 h. c and d, Nomarski interference micrographs of FPHL-1 cells, c, control; d, MP-treated cells. Rod-shaped mitochondria (arrows) are present in the perinuclear area of the untreated control cells, e and f, TEM of thin sections cut parallel to the growth surface of the liver cells, FNRL-1. e, control cells containing rod-like mitochondria and only a few lysosomes and residual bodies (arrow); I, MP- treated cells containing mostly ovoid mitochondria (M) and numerous lamellar bodies (L) in the cytoplasm. Fig. 2. a, TEM of thin sections from FNRL-1 cells treated with MP for 6 h, showing pleomorphic lamellar bodies, rough endoplasmic reticulum, and mitochondria. Multiple small lamellar bodies enclosed by a double membrane (arrows) are often seen. Lamellar bodies are sometimes seen associated with lysosomal structures (white arrow), b, TEM of thin sections from FNRL-1 cells treated with MP for 24 h, showing numerous lamellar bodies (L) with distinct lamellae. Ovoid mitochondria (M) and rough endoplasmic reticulum are also seen, c, whole mount of untreated FPHL-1 cells. Note the large number of very long mitochondria. As with the thin sections (Fig. 1e), there are hardly any lamellar bodies in this preparation, d, whole mount of FPHL-1 cells treated for 2 h with MP. Pleomorphic mitochondria and a few small osmiophilic lamellar bodies (arrows) are seen in the cytoplasm, e, whole mount of FPHL-1 cells after 24 h of MP treatment. Numerous large osmiophilic lamellar bodies (white arrows) of various sizes are seen in the perinuclear area. Inset, lamellar bodies at higher magnification. Bizarre-shaped mitochondria (black arrows) are sometimes seen in the treated cells, f, whole mount of FPHL-1 cells pretreated for 24 h with MP (25 ^g/ml; similar to Fig. 2e) and then grown for an additional 24 h in the absence of MP. The cells have lost most of the lamellar bodies. Mitochondrial morphology resembles that of the untreated control cells in Fig. 2c, except for a few bizarre ones (arrows).

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P. Thomas Iype, Corazon D. Bucana and Susan P. Kelley

Cancer Res 1985;45:2184-2191.

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