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Effect of carcinogenic polycyclic aromatic hydrocarbons on mouse embryonic cells in culture: Induction of spindle-shaped cells
Toxicology, 17 (1980) 39--49 © Elsevier/North-Holland Scientific Publishers Ltd.
EFFECT OF CARCINOGENIC POLYCYCLIC AROMATIC HYDROCARBONS ON MOUSE EMBRYONIC CELLS IN CULTURE: INDUCTION OF SPINDLE-SHAPED CELLS
HIROYA KAMEI Central Research Institute, The Japan Tobacco and Salt Public Corporation, Umegaoka, Midori-ku, Yokohama, Kanagawa, 227 (Japan) (Received May 28th, 1980) (Revision received July 14th, 1980) (Accepted July 21st, 1980)
6--2
SUMMARY
In cultured mouse embryonic cells (MECs) treated with benzo[a] pyrene (B[a]P), there appeared unusual type of fibroblasts, spindle-shaped cells (SP cells), which were characterized by their narrow bipolar shape, long cellular processes and optically distinct cell borders. Appearance of SP cells was massive and irreversible. The amount of SP cells increased with increasing concentrations of B[a]P, while early cytotoxicity did not. In various polycychc aromatic hydrocarbons (PAHs) tested, only potent carcinogens { 7,12-dimethylbenz[a]anthracene (DMBA), 3-methylcholanthrene (MCA), B[a] P, and dibenz[a,e]pyrene (DB[a,e]P)}induced SP cells. Among them, PAH having higher Iball's index induced SP cells at lower concentration and at an earlier time. Weak or non-carcinogenic PAHs including 3-hydroxybenzo[a]pyrene (3-OH-B[a]P) did not induce SP cells. ~-Napthoflavon (~NF) suppressed the induction of SP cell by carcinogenic PAH. SP cells did not appear spontaneously under various abnormal culture conditions. These results indicate that carcinogenic PAHs induce the appearance of a specific type of fibroblast, SP cells in MEC cultures in accordance with their carcinogenicity. Abbreviations: MEC, mouse embryonic cell;SP cell,spindle-shapedcell;PAH, polycyclic aromatic hydrocarbon; B[a ]P, benzo[a ]pyrene; M C A , 3-methylcholanthrene; D M B A , 7,12-dimethylbenz [a ]anthracene; D B [a,e]P, dibenz [a,e]pyrene; D B [a,h ]A, dibenz [a,h ]anthracene; BA, benz [a ]anthracene; B [e ]P, benzo [e ]pyrene; CR, chrysene; D B [de, kl ]A, dibenz[de, k/]anthracene; FL, fluoranthene; PY, pyrene; 3-OH-B[a]P, 3-hydroxybenzo[a]pyrene; aNF, ~-naphthoflavon; D M S O , dimethylsulphoxide; AHH, arylhydrocarbon hydroxylase; cAMP, adenosine 3",5'-cyclicmonophosphate; HEPES, N-2-hydroxyethylpiperazine-N~-2-ethanesulfonic acid;4- or 6-NQO, 4- or 6-nitroquinoline-l-oxide;M N N G ,
N-methyl-N'-nitro-N-nitrosoguanidine.
39
INTRODUCTION
It has been observed that SP cells or fusiform cells have appeared in mass culture of embryonic or lung cells treated with various chemical carcinogens in vitro [1--5]. Berwald and Sachs [1] reported that slender, spindle-shaped cells appeared in a culture of hamster embryonic cells treated with B[a] P or MCA within a few days. Kamahora and Kakunaga [2] observed a similar phenomenon with 4-nitroquinoline-l-oxide (4-NQO) but not with 6-nitroquinoline-l-oxide (6-NQO), which is a non-carcinogenic analogue of 4-NQO. Kuroki and Sato [3] also observed that 4-NQO and 2 other carcinogenic derivatives evoked this phenomenon but 2 non-carcinogenic derivatives of 4-NQO did not, and they called this phenomenon "early change". Similar "early changes" were reported with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) [4] and sodium nitrite [5]. Appearance of SP cells seems to be specific for the culture treated with chemical carcinogens and a useful marker for the early detection of carcinogens by mammalian cells in vitro. However, the phenomenon has not been investigated well. It is not clear whether the induction of SP cells corresponds to the carcinogenicity or to the cytotoxicity of the chemicals used. There is a possibility that the appearance of SP cells is an artifact caused by rapidly changed culture conditions [6]. Further, morphology of fibroblasts is alterable to an elongated form which resembles SP cells by various chemicals [7--11]. Thus, specificity of the appearance of SP cells in carcinogen-treated cultures is not clear. I have studied the induction of SP cells in mass culture of )'~EC by PAH. In this paper, results of investigations on the difference of SP cells from normal fibroblasts, kinetics of induction of SP cells by B[a] P, relation of the appearance of SP cells to the carcinogenicity of PAHs, and the absence of spontaneous appearance of SP cells under various culture conditions, are presented. The method for quantitative determination of the amount of SP cells is also presented.
MATERIALS AND METHODS
Chemicals 3-OH-B[a]P was provided by Dr. N. Kinoshita (the Univ. of Kyushu, Japan). Other PAHs (B[a] P, DMBA, MCA, DB[a,e] P, dibenz[a,h] anthracene (DB[a,h] A), benz[a] anthracene (BA), benzo[e] pyrene (B[e] P), chrysene (CR), dibenz[de, kl]anthracene (DB[de, kl]A), fluoranthene (FL), and pyrene (PY)) and aNF were obtained commercially. They were dissolved in DMSO and diluted with culture medium. All operations with PAHs were done avoiding artificial light.
Cell culture MECs were obtained from whole embryo of ICR mouse (at late gestation) by mincing and trypsinization (0.25% trypsin in Hank's solution, 37°C, 30 min). Tissue fragments were removed with filtration through gauze. Cells 40
were cultured in McCoy's 5A medium supplemented with 10% heat-inactivated calf serum or fetal calf serum and antibiotics (100 #g streptomycin and 100 units penicillin/ml) at 37°C in 5% CO2-alr. Secondary ~4_EC cultures were prepared from confluent primary cultures by trypsinization (0.125% trypsin) and plated at 3 × 10 s cells in 3 ml culture medium into 35-mm plastic Petri dish (Falcon plastics, CA, USA).
Treatments When secondary MEC cultures reached near confluence in 3--4 days after plating, medium was replaced with PAH-containing medium (0 day) and culture was continued for 24 h (or, in certain experiments, 2 days). After the treatment, PAH was removed by 2--3 changes of medium and cells were cultured in normal medium again. The medium was changed every 3 or 4 days thereafter. For continuous treatment, the medium containing the same dose of PAH was used. Ranges of concentrations of PAHs applied are listed in Table II. In some cases, a N F was added simultaneously with PAH or after the 24-h t r e a t m e n t with PAH. Control cultures were treated with the same concentrations of DMSO. Final concentrations of DMSO were usually 0.16 to 0.34%. When high doses of PAHs were applied, 1.6 or 2.0% DMSO was used. To examine the effect of changing the pH of the medium, medium was replaced with HEPES-buffered medium adjusted to adequate pH and cells were cultured in humidified air. After incubation in the first pH-medium for 24 h, the medium was changed to a second pH-medium. The medium was changed thereafter every day with the second pH-medium. Some cultures were buffered at the same pH throughout. For positive control, B[a]P was added to the first pH medium at a concentration of 10 pM. Assays Treated cells were observed for the appearance of SP cells under phase contrast microscope for 8 days or more. Bipolar narrow fibroblastic cells with single long cellular process on one or both ends of their bodies and optically distinct cell borders were determined as SP cells. For the quantitative determination of the n u m b e r of SP cells and total cells, cultures were fixed at an indicated time with methanol, stained with Giemsa's solution, water added, and observed under phase contrast microscope. The n u m b e r of SP cells and total cells present in 4 areas (0.024 m m 2 × 4) of a dish were counted. Each value is the mean of 2 dishes. Sizes of SP cells and normal fibroblasts were measured on microscopic photographs of living cells. The length of cellular process was also measured on fixed and stained samples under phase contrast microscopy. RESULTS
Appearance o f SP cells in B[a] P-treated 2IEC cultures and their difference from normal fibroblasts In B[a]P-treated MEC cultures, m a n y fibroblasts became narrower and 41
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Fig. 1. Time course of appearance of SP cells (A, B) and change in total cell number (C) in MEC cultures after treatment with B[a]P. MECs were treated with B[a]P at the concentrations of 0, 1.0, or 10 /~M for 24 h (0--1 day) and then cultured in normal medium without B[a ] P. Mean of 2 dishes, c o : 0 ~M B [a ] P-treated cultures. ~ ®: 1.0 uM B[a]P-treated cultures. • a: 10 uM B[a]P-treated cultures. *: Increase in total cell number compared to the value at 0 day. E~ : Duration of treatment with
B[a]P. t h e n masses o f SP cells a p p e a r e d w i t h i n 2--3 days. Percentages o f SP cells to t o t a l cells were 0 . 0 - - 0 . 6 f o r n o n - t r e a t e d cultures (at 0 d a y ) , 4 . 6 - - 2 9 . 1 f o r 10 pM B[a] P - t r e a t e d cultures (at 3 days), a n d 0 . 0 - - 0 . 7 f o r D M S O - t r e a t e d c o n t r o l cultures (at 3 days). Cells w h i c h h a d i n t e r m e d i a t e m o r p h o l o g y o f SP cells a n d n o r m a l bipolar fibroblasts were seen during 1--2 days. T h e y w e r e n o t c o u n t e d as SP cells. In a representative e x p e r i m e n t s h o w n in Fig. 1, t h e n u m b e r s o f SP cells/mm2 in 10 /~M B [ a ] P - t r e a t e d cultures were 0 at 0 d a y , 40 a t 1 d a y , a n d 2 0 9 0 a t 2 days (1 d a y a f t e r r e m o v a l o f B [ a ] P ) . SP cells a p p e a r e d w i t h o u t the c o n t i n u o u s presence o f B [ a ] P a n d increased rapidly f r o m 1 t o 2 d a y s ; a b o u t a 52-fold increase w i t h i n 24 h. T o t a l cell n u m b e r was slightly increased in the B [ a ] P-treated cultures at 1 day a n d d e c r e a s e d during 1--4 days. Figure 2 a n d Table I s h o w differences in m o r p h o l o g y o f SP cells in B[a] Pt r e a t e d cultures a n d n o r m a l bipolar fibroblasts in same-aged c o n t r o l cultures. SP cells were c h a r a c t e r i z e d by their long cellular process. T h e length o f t h e process was variable; 1 4 - - 1 6 0 p m . On the o t h e r h a n d , n o r m a l fibroblasts w i t h cellular processes were difficult to define. SP cells h a d n a r r o w and
42
Fig. 2. SP cells in B [ a ] P - t r e a t e d MEC culture and n o r m a l fibroblasts in same aged c o n t r o l MEC culture. MECs w e r e t r e a t e d w i t h B [ a ] P at a c o n c e n t r a t i o n o f 10 ~M or 0.16% DMSO f o r 24 h and t h e n c u l t u r e d in n o r m a l m e d i u m w i t h o u t B [ a ] P for 2 days. A: B [ a ] P - t r e a t e d MEC culture. A r r o w s indicate t h e SP cells. B: DMSO-treated c o n t r o l MEC culture. Phase c o n t r a s t . × 400.
43
TABLE I SIZES O F SP C E L L S IN B [ a ] P - T R E A T E D M E C C U L T U R E S A N D N O R M A L F I B R O B L A S T S IN S A M E - A G E D C O N T R O L M E C C U L T U R E S Exp. a
A B C
Cellsb
SP cells Fibroblasts SP cells " Fibroblasts SP cells
BIPOLAR
Width of cellbodies
Length of cell bodies Length of process
Mean
S.D.
Range
(~m)
(urn)
6.9" 11.8 6.4" 9.7 NDd
0.9 3.0 0.9 1.9
No. c
No.
(~m) 62 50 42 43
31--62 __e 38--60 -ND
Range
No.
(~m) 14
14--115
15
11
20--106
14
15--160
29
aA; Cultured in normal medium for 2 days after 24-h treatment with B[a]P at a concentration of 10 uM or 0.16% DMSO. Measured on phase contrast microscopic photographs of living cells. B; Cultured in normal medium for 5 days after 2-day treatment with B[a]P at a concentration of 10 ~M or 1.6% DMSO. Measured as in A. C; Cultured in normal medium for 2 days after 24-h treatment with B[a]P at a concentration of 10 uM. Measured directly on fixed and stained samples by measuring equipment of phase contrast microscope. bSp cells; in B[a]P-treated MEC culture. Fibroblasts; Bipolar fibroblasts in DMSOtreated control MEC culture. CNo. of cells measured. dNot determined. eDifficult to measure. *Significantly different from the value of control fibroblasts (P < 0.01 ).
dense cell bodies in c o n t r a s t t o t h e w i d e a n d flat a p p e a r a n c e o f n o r m a l fibroblasts. T h e w i d t h o f t h e cell b o d i e s o f SP cells was significantly differe n t in its d i s t r i b u t i o n a n d m e a n f r o m t h a t o f n o r m a l fibroblasts (P < 0.01). SP cells a r r a n g e d m a i n l y parallel, b u t in s o m e cases or in s o m e areas o f c u l t u r e s t h e y a r r a n g e d criss-cross. T h e y grew o n wide-spread cells a n d a l o n g t h e colonies o f epitherial cells. F i b r o b l a s t s in c o n t r o l cultures a r r a n g e d a n d grew a l m o s t in the same w a y as SP cells. W h e n B [ a ] P-treated cells were r e p l a t e d , SP cells c o n t i n u e d t o be p r e s e n t in the fresh cultures. T h e characteristic m o r p h o l o g y o f SP cells was mainr a i n e d at a n y cell d e n s i t y a f t e r replating. N o SP cell was o b s e r v e d in t h e r e p l a t e d c u l t u r e s o f c o n t r o l cells.
Dose response for appearance o f SP cells and cytotoxicity by B[a] P Figure 3 s h o w s the n u m b e r s o f SP cells a n d t o t a l cells in MEC cultures t r e a t e d w i t h various c o n c e n t r a t i o n s o f B [ a ] P . SP cells a p p e a r e d a b o v e 1.0 pM B [ a ] P. T h e a m o u n t o f SP cells increased progressively w i t h increasing c o n c e n t r a t i o n s o f B [ a ] P f r o m 1.0 t o 20 pM. Decrease in t o t a l cell n u m b e r also o c c u r r e d a b o v e 1.0 ~,'I B [ a ] P. But, t h e degree o f the decrease was a l m o s t the same at a n y c o n c e n t r a t i o n s o f B [ a ] P f r o m 1.0 pM to 20 ~ J .
44
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Fig. 3. Dose r e s p o n s e for a p p e a r a n c e o f SP cells (A, B) and change in t o t a l cell n u m b e r
(C) in ME C cultures by B[a ]P. MECs were treated with B[a]P at various concentrations indicated in t h e figure for 24 h and then cultured in normal medium without B[a]P for 2 days. M e a n o f 2 dishes. * : Increase in t o t a l cell n u m b e r c o m p a r e d t o t h e value at 0 day.
Induction o f SP cells by various PAils Table II shows abilities of various PAHs to induce SP cells and their carcinogenicity expressed as Iball's index [ 1 2 , 1 3 ] . Among the PAHs tested, DMBA, MCA, B[a]P, and D B [ a , e ] P induced SP cells. These chemicals are p o t e n t carcinogens and have Iball's indices of 50 and over. SP cells appeared in 2--3 days in the cultures treated with DMBA, MCA, or B [a] P, and in 4--7 days with DB[a,e] P. The lowest concentrations effective to induce SP cells were 0.1 pM for DMBA, 1.0 uM for MCA and B[a]P, and 20 pM_ for DB[a,e] P. The SP cells induced by these PAHs appeared in large numbers and did n o t disappear in the replated cultures. Degradations of some cells were also observed in the cultures treated with the carcinogenic PAHs at the doses at which induction of SP cells occurred. In contrast to the above results, DB[a,h] A, BA, B[e] P, CR, DB[de, hl] A, FL, or PY did n o t induce SP cells at any concentrations used. These chemicals are weak or non-carcinogens and have Iball's indices of 26 and less. Among them, PY {above 500 uM) and FL {above 1000Uh~) caused significant
45
TABLE II ABILITIES OF VARIOUS PAHS TO INDUCE SP CELLS IN MEC CULTURE Treatmenta
SP cell inductionc
Iball's index g
PAH
Concentrationb (#M)
Inductiond
Lowest Time of concentration appearance positive e (Day)f (~M)
DMBA MCA B[a]P DB[a,e]P DB[a,h]A BA B[e]P CR DB[de, kl]A FL PY 3-OH-B[a]P
0.0036-2.6 0.11 -- 59 0.10 -- 100 1.1 -- 147" 0.33 -- 370* 1.4 --2000* 0.36 -- 213" 0.14 -- 225* 0.36 -- 149" 160 --2000* 160 --2000* 0.23 230
+ + + + -
0.1 1.0 1.0 20
-
-
-
2 or 3 2 or 3 2or3 4to7
96, 151 80, 93 72 50 26 5 3 3 0 0 0
-
aMedium was replaced with PAH-containing medium and cells were cultured for 24 h. After the treatment, cells were cultured in normal medium without PAH. In experiments for DB[a,e]P, DB[a,h]A, FL, and PY, cells were also treated for 2 days, and for 3-OH-B[a]P, cells were also treated continuously, all of which rendered essentially the same results as in the table. bOnly lowest and highest concentrations applied are listed. Concentration of each PAH was varied in half-log scale. eInduction of SP cells within 8 days. d+ ; SP cells were induced. --; No SP cell was induced. eSp cells were induced in MEC cultures treated at or above this concentration. fDays after replacement with PAH-containing medium. gFrom Iball [12] and Arcos and Argus [13]. *Highest dose appliable due to the limited solubility of the chemical in DMSO or in the final culture medium. cytotoxic effects including shrinkage and death of some fibroblasts without i n d u c i n g SP cells. O t h e r c h e m i c a l s h a d n o a p p r e c i a b l e e f f e c t o n t h e f i b r o b l a s t s e v e n a t t h e h i g h e s t d o s e s t e s t e d . SP cells w e r e i n d u c e d b y B [ a ] P ( 1 0 u M ) a p p l i e d w i t h 1 . 6 o r 2 . 0 % D M S O ( T a b l e I, e x p . B). T h e s e c o n c e n t r a tions of DMSO were used to attain high doses of PAHs. 3 - O H - B [ a ] P h a d n o a p p r e c i a b l e e f f e c t o n t h e cells u p t o 3 0 p M e v e n w h e n applied continuously. Above 40 #M, 3-OH-B[a]P caused partial regression a n d d e t a c h m e n t o f t h e cells. T h e s e e f f e c t s w e r e o b v i o u s b y 6 h a f t e r t h e a d d i t i o n o f 3 - O H - B [ a ] P. S o m e n a r r o w cells w e r e o b s e r v e d t e m p o r a r i l y i n t h e c o u r s e o f cell r e g r e s s i o n . B u t a f t e r r e m o v a l o f 3 - O H - B [a] P, s u r v i v i n g cells retained their expanded state and began to grow again. Above 100 pM 3 - O H - B [ a ] P, m a n y cells b e c a m e s p h e r i c a l a n d d e a d . N o SP cells w e r e i n d u c e d b y a n y dose level a n d after a n y d u r a t i o n of t r e a t m e n t of 3 - O H - B [ a ] P .
46
Effects o f aNF on the induction o f SP cells by PAHs Induction of SP cells by DMBA, MCA, or B[a] P was suppressed when a N F was added simultaneously and at the same concentration with the PAH. But when a N F was added after 24 h treatment of the PAH, it was not suppressive, a N F per se had no effect on the cells up to 50 p~.,'I. Effects o f altered culture conditions Very few SP cells appeared in the cultures in which the pH was rapidly changed from 6.0 to 8.0, 8.0 to 6.6, or 8.7 to 6.6 except when B [ a ] P was added. In pH-fixed experiment, SP cells appeared at pH 7.0, 8.0, or 8.7 when B[a] P was added. Changing the temperature from (a) 37°C to 40°C, or (b) 37°C to 4°C and then to 37°C, did n o t result in SP cell appearance. MEC cultures brought to 4°C became shrunken leaving long fine projections on their sides, but recovered to their normal morphology when returned to 37°C. Increasing the osmolarity by adding NaC1 also failed to induce SP cells. Greater than 0.7% NaC1 was toxic to MEC cultures. Changing the serum concentration in the medium from 10% to 1.0 or 0.3% caused suppression of cell growth b u t did n o t induce SP cells. They were induced when B[a] P was added at a concentration of 10 pM to MEC cultured at such low serum concentrations.
DISCUSSION
B[a] P and other carcinogenic PAHs induced the appearance of SP cells in mass cultures of MEC within a few days. The morphology of SP cells was clearly different from normal bipolar fibroblasts. They were stable and unaffected by cell densities in contrast to alterable morphology of normal fibroblasts. SP cells were n o t the normally accumulating aged cells. The SP cells induced by B [ a ] P may be the fibroblasts altered in their shape and n o t the selectively survived and increased SP-like cells already present in few numbers before B[a]P-treatment. This is suggested by the following: (a) The mass of SP cells appeared suddenly at 2 days after the B [ a ] P addition. The number of SP cells increased 52-fold from 1 to 2 days in 10 pM B[a] P-treated culture (Fig. 1). This 52-fold increase requires 6 cell divisions within 24 h, which would n o t normally occur. (b) Intermediate narrow cells were observed preceding the appearance of SP cells. (c) Arrangement of SP cells and their relation to epitherial colonies were almost the same with those of fibroblasts in control cultures. Fibroblasts are known to alter their morphology to elongated form in the presence of cAMP or other chemicals [7--11]. But, the induction of SP cells in MEC cultures by B [ a ] P is different from this. Alteration induced with cAMP or others occurs within a few hours after addition and is reversible when the inducing agent is removed from the culture medium [8,10]. On the other hand, induction of SP cells by B [ a ] P is delayed for 2 days after the addition of B[a] P. It was not reversible after the removal
47
of B[a] P. SP cells were maintained even after replating them to a fresh dish with fresh culture medium. Induction of SP cells in MEC by PAH is n o t a non-specific artifact. It did n o t occur in response to changes of pH of the medium as described by DiPaolo and Donovan [6]. SP cells did n o t appear spontaneously under abnormal or unsuitable culture conditions in temperature, osmolarity, or serum concentration. Suppression of cell growth and degradation of some cells were observed c o n c o m i t a n t l y with the appearance of SP cells. But these early cytotoxicities did n o t relate to the induction of SP cells, as both showed different dose-responses to B[a] P (Fig. 3). In various PAHs, only p o t e n t carcinogens (DMBA, MCA, B[a]P, and DB[a,e] P) induced SP cells in MEC cultures. Among these PAHs, those with the higher IbaU's indices [12,13] induced SP cells at lower dose and in a shorter time (Table II). Thus activities of PAHs to induce SP cells corresponded to the carcinogenicities of the chemicals, a N F is known as a p o t e n t inhibitor of arylhydrocarbon hydroxylase (AHH) [14]. As a N F suppressed the induction of SP cells by PAH, metabolism of PAH by AHH m a y be a prerequisite for the induction. This is further suggested by the fact t h a t when added after 24 h exposure to PAH, a N F did n o t prevent induction. DB[a,h] A, which has Iball's index o f 26, did n o t induce SP cells. It may be possible that the doses and/or its metabolic activation by the cells were insufficient for induction. Results obtained by 3-OH-B[a]P are interesting as 3-OH-B[a]P is a representative cytotoxic metabolite of B[a] P by AHH [15] which has no carcinogenic properties [16]. The main effects of 3-OH-B[a]P on HECs were regression and detachment of the cells, which were rapid and partially reversible, and n o t the induction of SP cells. Active metabolite(s) of B[a] P for the induction may be different from 3-OH-B[a] P. In conclusion, the appearance of SP cells in MEC cultures treated with PAH corresponds to the carcinogenicity of the chemicals. SP cells may be fibroblasts altered in their morphology irreversibly by the treatment with carcinogens. Appearance of SP cells may reflect some c o m m o n action of carcinogens on the cells and may be useful for rapid detection of carcinogens, though further studies are required. ACKNOWLEDGEMENT
I t h a n k Dr. N. Kinoshita (Medical and Clinical College, the Univ. of Kyushu) for providing 3-OH-B[a] P. REFERENCES
1 2 3 4
48
Y. Berwald and L. Sachs, J. Natl. Cancer Inst., 35 (1965) 641. J. Kamahora and T. Kakunaga, Biken J., 9 (1966) 295. T. Kuroki and H. Sato, J. Natl. Cancer Inst., 41 (1968) 53. N. Inui, S. Takayama and T. Sugimura, J. Natl. Cancer Inst., 48 (1972) 1409.
5 H. Tsuda, N. Inui and S. Takayama, Biochem. Biophys. Res. Commun., 55 (1973) 1117. 6 J.A. DiPaolo and P.J. Donovan, Exp. Cell Res., 48 (1967) 361. 7 R.P. Cox and B.M. Gesner, Proc. Natl. Acad. Sci. USA, 54 (1965) 1571. 8 G.S. Johnson, R.M. Friedman and I. Pastan, Proc. Natl. Acad. Sci. USA, 68 (1971) 425. 9 G.S. Johnson and I. Pastan, J. Natl. Cancer Inst., 47 (1971) 1357. 10 L. Diamond, S. O'Brien, C. Donaldson and Y. Shimizu, Int. J. Cancer, 13 (1974) 721. 11 G.S. Johnson and J.P. Schwartz, Exp. Cell Res., 97 (1976) 281. 12 J. Iball, Am. J. Cancer, 35 (1939) 188. 13 J.C. Arcos and M.F. Argus, Adv. Cancer Res., 11 (1968) 305. 14 L. Diamond and H.V. Gelboin, Science, 166 (1969) 1023. 15 H.V. Gelboin, E. Huberman and L. Sachs, Proc. Natl. Acad. Sci. USA, 64 (1969) 1188. 16 P.G. Wislocki, R.L. Chang, A.W. Wood, W. Levin, H. Yagi, O. Hernandez, H.D. Mah, P.M. Dansette, D.M. Jerina and A.H. Conney, Cancer Res., 37 (1977) 2608.
49
EFFECT OF CARCINOGENIC POLYCYCLIC AROMATIC HYDROCARBONS ON MOUSE EMBRYONIC CELLS IN CULTURE: INDUCTION OF SPINDLE-SHAPED CELLS
HIROYA KAMEI Central Research Institute, The Japan Tobacco and Salt Public Corporation, Umegaoka, Midori-ku, Yokohama, Kanagawa, 227 (Japan) (Received May 28th, 1980) (Revision received July 14th, 1980) (Accepted July 21st, 1980)
6--2
SUMMARY
In cultured mouse embryonic cells (MECs) treated with benzo[a] pyrene (B[a]P), there appeared unusual type of fibroblasts, spindle-shaped cells (SP cells), which were characterized by their narrow bipolar shape, long cellular processes and optically distinct cell borders. Appearance of SP cells was massive and irreversible. The amount of SP cells increased with increasing concentrations of B[a]P, while early cytotoxicity did not. In various polycychc aromatic hydrocarbons (PAHs) tested, only potent carcinogens { 7,12-dimethylbenz[a]anthracene (DMBA), 3-methylcholanthrene (MCA), B[a] P, and dibenz[a,e]pyrene (DB[a,e]P)}induced SP cells. Among them, PAH having higher Iball's index induced SP cells at lower concentration and at an earlier time. Weak or non-carcinogenic PAHs including 3-hydroxybenzo[a]pyrene (3-OH-B[a]P) did not induce SP cells. ~-Napthoflavon (~NF) suppressed the induction of SP cell by carcinogenic PAH. SP cells did not appear spontaneously under various abnormal culture conditions. These results indicate that carcinogenic PAHs induce the appearance of a specific type of fibroblast, SP cells in MEC cultures in accordance with their carcinogenicity. Abbreviations: MEC, mouse embryonic cell;SP cell,spindle-shapedcell;PAH, polycyclic aromatic hydrocarbon; B[a ]P, benzo[a ]pyrene; M C A , 3-methylcholanthrene; D M B A , 7,12-dimethylbenz [a ]anthracene; D B [a,e]P, dibenz [a,e]pyrene; D B [a,h ]A, dibenz [a,h ]anthracene; BA, benz [a ]anthracene; B [e ]P, benzo [e ]pyrene; CR, chrysene; D B [de, kl ]A, dibenz[de, k/]anthracene; FL, fluoranthene; PY, pyrene; 3-OH-B[a]P, 3-hydroxybenzo[a]pyrene; aNF, ~-naphthoflavon; D M S O , dimethylsulphoxide; AHH, arylhydrocarbon hydroxylase; cAMP, adenosine 3",5'-cyclicmonophosphate; HEPES, N-2-hydroxyethylpiperazine-N~-2-ethanesulfonic acid;4- or 6-NQO, 4- or 6-nitroquinoline-l-oxide;M N N G ,
N-methyl-N'-nitro-N-nitrosoguanidine.
39
INTRODUCTION
It has been observed that SP cells or fusiform cells have appeared in mass culture of embryonic or lung cells treated with various chemical carcinogens in vitro [1--5]. Berwald and Sachs [1] reported that slender, spindle-shaped cells appeared in a culture of hamster embryonic cells treated with B[a] P or MCA within a few days. Kamahora and Kakunaga [2] observed a similar phenomenon with 4-nitroquinoline-l-oxide (4-NQO) but not with 6-nitroquinoline-l-oxide (6-NQO), which is a non-carcinogenic analogue of 4-NQO. Kuroki and Sato [3] also observed that 4-NQO and 2 other carcinogenic derivatives evoked this phenomenon but 2 non-carcinogenic derivatives of 4-NQO did not, and they called this phenomenon "early change". Similar "early changes" were reported with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) [4] and sodium nitrite [5]. Appearance of SP cells seems to be specific for the culture treated with chemical carcinogens and a useful marker for the early detection of carcinogens by mammalian cells in vitro. However, the phenomenon has not been investigated well. It is not clear whether the induction of SP cells corresponds to the carcinogenicity or to the cytotoxicity of the chemicals used. There is a possibility that the appearance of SP cells is an artifact caused by rapidly changed culture conditions [6]. Further, morphology of fibroblasts is alterable to an elongated form which resembles SP cells by various chemicals [7--11]. Thus, specificity of the appearance of SP cells in carcinogen-treated cultures is not clear. I have studied the induction of SP cells in mass culture of )'~EC by PAH. In this paper, results of investigations on the difference of SP cells from normal fibroblasts, kinetics of induction of SP cells by B[a] P, relation of the appearance of SP cells to the carcinogenicity of PAHs, and the absence of spontaneous appearance of SP cells under various culture conditions, are presented. The method for quantitative determination of the amount of SP cells is also presented.
MATERIALS AND METHODS
Chemicals 3-OH-B[a]P was provided by Dr. N. Kinoshita (the Univ. of Kyushu, Japan). Other PAHs (B[a] P, DMBA, MCA, DB[a,e] P, dibenz[a,h] anthracene (DB[a,h] A), benz[a] anthracene (BA), benzo[e] pyrene (B[e] P), chrysene (CR), dibenz[de, kl]anthracene (DB[de, kl]A), fluoranthene (FL), and pyrene (PY)) and aNF were obtained commercially. They were dissolved in DMSO and diluted with culture medium. All operations with PAHs were done avoiding artificial light.
Cell culture MECs were obtained from whole embryo of ICR mouse (at late gestation) by mincing and trypsinization (0.25% trypsin in Hank's solution, 37°C, 30 min). Tissue fragments were removed with filtration through gauze. Cells 40
were cultured in McCoy's 5A medium supplemented with 10% heat-inactivated calf serum or fetal calf serum and antibiotics (100 #g streptomycin and 100 units penicillin/ml) at 37°C in 5% CO2-alr. Secondary ~4_EC cultures were prepared from confluent primary cultures by trypsinization (0.125% trypsin) and plated at 3 × 10 s cells in 3 ml culture medium into 35-mm plastic Petri dish (Falcon plastics, CA, USA).
Treatments When secondary MEC cultures reached near confluence in 3--4 days after plating, medium was replaced with PAH-containing medium (0 day) and culture was continued for 24 h (or, in certain experiments, 2 days). After the treatment, PAH was removed by 2--3 changes of medium and cells were cultured in normal medium again. The medium was changed every 3 or 4 days thereafter. For continuous treatment, the medium containing the same dose of PAH was used. Ranges of concentrations of PAHs applied are listed in Table II. In some cases, a N F was added simultaneously with PAH or after the 24-h t r e a t m e n t with PAH. Control cultures were treated with the same concentrations of DMSO. Final concentrations of DMSO were usually 0.16 to 0.34%. When high doses of PAHs were applied, 1.6 or 2.0% DMSO was used. To examine the effect of changing the pH of the medium, medium was replaced with HEPES-buffered medium adjusted to adequate pH and cells were cultured in humidified air. After incubation in the first pH-medium for 24 h, the medium was changed to a second pH-medium. The medium was changed thereafter every day with the second pH-medium. Some cultures were buffered at the same pH throughout. For positive control, B[a]P was added to the first pH medium at a concentration of 10 pM. Assays Treated cells were observed for the appearance of SP cells under phase contrast microscope for 8 days or more. Bipolar narrow fibroblastic cells with single long cellular process on one or both ends of their bodies and optically distinct cell borders were determined as SP cells. For the quantitative determination of the n u m b e r of SP cells and total cells, cultures were fixed at an indicated time with methanol, stained with Giemsa's solution, water added, and observed under phase contrast microscope. The n u m b e r of SP cells and total cells present in 4 areas (0.024 m m 2 × 4) of a dish were counted. Each value is the mean of 2 dishes. Sizes of SP cells and normal fibroblasts were measured on microscopic photographs of living cells. The length of cellular process was also measured on fixed and stained samples under phase contrast microscopy. RESULTS
Appearance o f SP cells in B[a] P-treated 2IEC cultures and their difference from normal fibroblasts In B[a]P-treated MEC cultures, m a n y fibroblasts became narrower and 41
0
2.0
% E
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4O
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0
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3
.4
6
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,~
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Fig. 1. Time course of appearance of SP cells (A, B) and change in total cell number (C) in MEC cultures after treatment with B[a]P. MECs were treated with B[a]P at the concentrations of 0, 1.0, or 10 /~M for 24 h (0--1 day) and then cultured in normal medium without B[a ] P. Mean of 2 dishes, c o : 0 ~M B [a ] P-treated cultures. ~ ®: 1.0 uM B[a]P-treated cultures. • a: 10 uM B[a]P-treated cultures. *: Increase in total cell number compared to the value at 0 day. E~ : Duration of treatment with
B[a]P. t h e n masses o f SP cells a p p e a r e d w i t h i n 2--3 days. Percentages o f SP cells to t o t a l cells were 0 . 0 - - 0 . 6 f o r n o n - t r e a t e d cultures (at 0 d a y ) , 4 . 6 - - 2 9 . 1 f o r 10 pM B[a] P - t r e a t e d cultures (at 3 days), a n d 0 . 0 - - 0 . 7 f o r D M S O - t r e a t e d c o n t r o l cultures (at 3 days). Cells w h i c h h a d i n t e r m e d i a t e m o r p h o l o g y o f SP cells a n d n o r m a l bipolar fibroblasts were seen during 1--2 days. T h e y w e r e n o t c o u n t e d as SP cells. In a representative e x p e r i m e n t s h o w n in Fig. 1, t h e n u m b e r s o f SP cells/mm2 in 10 /~M B [ a ] P - t r e a t e d cultures were 0 at 0 d a y , 40 a t 1 d a y , a n d 2 0 9 0 a t 2 days (1 d a y a f t e r r e m o v a l o f B [ a ] P ) . SP cells a p p e a r e d w i t h o u t the c o n t i n u o u s presence o f B [ a ] P a n d increased rapidly f r o m 1 t o 2 d a y s ; a b o u t a 52-fold increase w i t h i n 24 h. T o t a l cell n u m b e r was slightly increased in the B [ a ] P-treated cultures at 1 day a n d d e c r e a s e d during 1--4 days. Figure 2 a n d Table I s h o w differences in m o r p h o l o g y o f SP cells in B[a] Pt r e a t e d cultures a n d n o r m a l bipolar fibroblasts in same-aged c o n t r o l cultures. SP cells were c h a r a c t e r i z e d by their long cellular process. T h e length o f t h e process was variable; 1 4 - - 1 6 0 p m . On the o t h e r h a n d , n o r m a l fibroblasts w i t h cellular processes were difficult to define. SP cells h a d n a r r o w and
42
Fig. 2. SP cells in B [ a ] P - t r e a t e d MEC culture and n o r m a l fibroblasts in same aged c o n t r o l MEC culture. MECs w e r e t r e a t e d w i t h B [ a ] P at a c o n c e n t r a t i o n o f 10 ~M or 0.16% DMSO f o r 24 h and t h e n c u l t u r e d in n o r m a l m e d i u m w i t h o u t B [ a ] P for 2 days. A: B [ a ] P - t r e a t e d MEC culture. A r r o w s indicate t h e SP cells. B: DMSO-treated c o n t r o l MEC culture. Phase c o n t r a s t . × 400.
43
TABLE I SIZES O F SP C E L L S IN B [ a ] P - T R E A T E D M E C C U L T U R E S A N D N O R M A L F I B R O B L A S T S IN S A M E - A G E D C O N T R O L M E C C U L T U R E S Exp. a
A B C
Cellsb
SP cells Fibroblasts SP cells " Fibroblasts SP cells
BIPOLAR
Width of cellbodies
Length of cell bodies Length of process
Mean
S.D.
Range
(~m)
(urn)
6.9" 11.8 6.4" 9.7 NDd
0.9 3.0 0.9 1.9
No. c
No.
(~m) 62 50 42 43
31--62 __e 38--60 -ND
Range
No.
(~m) 14
14--115
15
11
20--106
14
15--160
29
aA; Cultured in normal medium for 2 days after 24-h treatment with B[a]P at a concentration of 10 uM or 0.16% DMSO. Measured on phase contrast microscopic photographs of living cells. B; Cultured in normal medium for 5 days after 2-day treatment with B[a]P at a concentration of 10 ~M or 1.6% DMSO. Measured as in A. C; Cultured in normal medium for 2 days after 24-h treatment with B[a]P at a concentration of 10 uM. Measured directly on fixed and stained samples by measuring equipment of phase contrast microscope. bSp cells; in B[a]P-treated MEC culture. Fibroblasts; Bipolar fibroblasts in DMSOtreated control MEC culture. CNo. of cells measured. dNot determined. eDifficult to measure. *Significantly different from the value of control fibroblasts (P < 0.01 ).
dense cell bodies in c o n t r a s t t o t h e w i d e a n d flat a p p e a r a n c e o f n o r m a l fibroblasts. T h e w i d t h o f t h e cell b o d i e s o f SP cells was significantly differe n t in its d i s t r i b u t i o n a n d m e a n f r o m t h a t o f n o r m a l fibroblasts (P < 0.01). SP cells a r r a n g e d m a i n l y parallel, b u t in s o m e cases or in s o m e areas o f c u l t u r e s t h e y a r r a n g e d criss-cross. T h e y grew o n wide-spread cells a n d a l o n g t h e colonies o f epitherial cells. F i b r o b l a s t s in c o n t r o l cultures a r r a n g e d a n d grew a l m o s t in the same w a y as SP cells. W h e n B [ a ] P-treated cells were r e p l a t e d , SP cells c o n t i n u e d t o be p r e s e n t in the fresh cultures. T h e characteristic m o r p h o l o g y o f SP cells was mainr a i n e d at a n y cell d e n s i t y a f t e r replating. N o SP cell was o b s e r v e d in t h e r e p l a t e d c u l t u r e s o f c o n t r o l cells.
Dose response for appearance o f SP cells and cytotoxicity by B[a] P Figure 3 s h o w s the n u m b e r s o f SP cells a n d t o t a l cells in MEC cultures t r e a t e d w i t h various c o n c e n t r a t i o n s o f B [ a ] P . SP cells a p p e a r e d a b o v e 1.0 pM B [ a ] P. T h e a m o u n t o f SP cells increased progressively w i t h increasing c o n c e n t r a t i o n s o f B [ a ] P f r o m 1.0 t o 20 pM. Decrease in t o t a l cell n u m b e r also o c c u r r e d a b o v e 1.0 ~,'I B [ a ] P. But, t h e degree o f the decrease was a l m o s t the same at a n y c o n c e n t r a t i o n s o f B [ a ] P f r o m 1.0 pM to 20 ~ J .
44
(y 0 >(
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0.2
I
2
10
20
0
-m0
Concentration of B(a)P, pM
Fig. 3. Dose r e s p o n s e for a p p e a r a n c e o f SP cells (A, B) and change in t o t a l cell n u m b e r
(C) in ME C cultures by B[a ]P. MECs were treated with B[a]P at various concentrations indicated in t h e figure for 24 h and then cultured in normal medium without B[a]P for 2 days. M e a n o f 2 dishes. * : Increase in t o t a l cell n u m b e r c o m p a r e d t o t h e value at 0 day.
Induction o f SP cells by various PAils Table II shows abilities of various PAHs to induce SP cells and their carcinogenicity expressed as Iball's index [ 1 2 , 1 3 ] . Among the PAHs tested, DMBA, MCA, B[a]P, and D B [ a , e ] P induced SP cells. These chemicals are p o t e n t carcinogens and have Iball's indices of 50 and over. SP cells appeared in 2--3 days in the cultures treated with DMBA, MCA, or B [a] P, and in 4--7 days with DB[a,e] P. The lowest concentrations effective to induce SP cells were 0.1 pM for DMBA, 1.0 uM for MCA and B[a]P, and 20 pM_ for DB[a,e] P. The SP cells induced by these PAHs appeared in large numbers and did n o t disappear in the replated cultures. Degradations of some cells were also observed in the cultures treated with the carcinogenic PAHs at the doses at which induction of SP cells occurred. In contrast to the above results, DB[a,h] A, BA, B[e] P, CR, DB[de, hl] A, FL, or PY did n o t induce SP cells at any concentrations used. These chemicals are weak or non-carcinogens and have Iball's indices of 26 and less. Among them, PY {above 500 uM) and FL {above 1000Uh~) caused significant
45
TABLE II ABILITIES OF VARIOUS PAHS TO INDUCE SP CELLS IN MEC CULTURE Treatmenta
SP cell inductionc
Iball's index g
PAH
Concentrationb (#M)
Inductiond
Lowest Time of concentration appearance positive e (Day)f (~M)
DMBA MCA B[a]P DB[a,e]P DB[a,h]A BA B[e]P CR DB[de, kl]A FL PY 3-OH-B[a]P
0.0036-2.6 0.11 -- 59 0.10 -- 100 1.1 -- 147" 0.33 -- 370* 1.4 --2000* 0.36 -- 213" 0.14 -- 225* 0.36 -- 149" 160 --2000* 160 --2000* 0.23 230
+ + + + -
0.1 1.0 1.0 20
-
-
-
2 or 3 2 or 3 2or3 4to7
96, 151 80, 93 72 50 26 5 3 3 0 0 0
-
aMedium was replaced with PAH-containing medium and cells were cultured for 24 h. After the treatment, cells were cultured in normal medium without PAH. In experiments for DB[a,e]P, DB[a,h]A, FL, and PY, cells were also treated for 2 days, and for 3-OH-B[a]P, cells were also treated continuously, all of which rendered essentially the same results as in the table. bOnly lowest and highest concentrations applied are listed. Concentration of each PAH was varied in half-log scale. eInduction of SP cells within 8 days. d+ ; SP cells were induced. --; No SP cell was induced. eSp cells were induced in MEC cultures treated at or above this concentration. fDays after replacement with PAH-containing medium. gFrom Iball [12] and Arcos and Argus [13]. *Highest dose appliable due to the limited solubility of the chemical in DMSO or in the final culture medium. cytotoxic effects including shrinkage and death of some fibroblasts without i n d u c i n g SP cells. O t h e r c h e m i c a l s h a d n o a p p r e c i a b l e e f f e c t o n t h e f i b r o b l a s t s e v e n a t t h e h i g h e s t d o s e s t e s t e d . SP cells w e r e i n d u c e d b y B [ a ] P ( 1 0 u M ) a p p l i e d w i t h 1 . 6 o r 2 . 0 % D M S O ( T a b l e I, e x p . B). T h e s e c o n c e n t r a tions of DMSO were used to attain high doses of PAHs. 3 - O H - B [ a ] P h a d n o a p p r e c i a b l e e f f e c t o n t h e cells u p t o 3 0 p M e v e n w h e n applied continuously. Above 40 #M, 3-OH-B[a]P caused partial regression a n d d e t a c h m e n t o f t h e cells. T h e s e e f f e c t s w e r e o b v i o u s b y 6 h a f t e r t h e a d d i t i o n o f 3 - O H - B [ a ] P. S o m e n a r r o w cells w e r e o b s e r v e d t e m p o r a r i l y i n t h e c o u r s e o f cell r e g r e s s i o n . B u t a f t e r r e m o v a l o f 3 - O H - B [a] P, s u r v i v i n g cells retained their expanded state and began to grow again. Above 100 pM 3 - O H - B [ a ] P, m a n y cells b e c a m e s p h e r i c a l a n d d e a d . N o SP cells w e r e i n d u c e d b y a n y dose level a n d after a n y d u r a t i o n of t r e a t m e n t of 3 - O H - B [ a ] P .
46
Effects o f aNF on the induction o f SP cells by PAHs Induction of SP cells by DMBA, MCA, or B[a] P was suppressed when a N F was added simultaneously and at the same concentration with the PAH. But when a N F was added after 24 h treatment of the PAH, it was not suppressive, a N F per se had no effect on the cells up to 50 p~.,'I. Effects o f altered culture conditions Very few SP cells appeared in the cultures in which the pH was rapidly changed from 6.0 to 8.0, 8.0 to 6.6, or 8.7 to 6.6 except when B [ a ] P was added. In pH-fixed experiment, SP cells appeared at pH 7.0, 8.0, or 8.7 when B[a] P was added. Changing the temperature from (a) 37°C to 40°C, or (b) 37°C to 4°C and then to 37°C, did n o t result in SP cell appearance. MEC cultures brought to 4°C became shrunken leaving long fine projections on their sides, but recovered to their normal morphology when returned to 37°C. Increasing the osmolarity by adding NaC1 also failed to induce SP cells. Greater than 0.7% NaC1 was toxic to MEC cultures. Changing the serum concentration in the medium from 10% to 1.0 or 0.3% caused suppression of cell growth b u t did n o t induce SP cells. They were induced when B[a] P was added at a concentration of 10 pM to MEC cultured at such low serum concentrations.
DISCUSSION
B[a] P and other carcinogenic PAHs induced the appearance of SP cells in mass cultures of MEC within a few days. The morphology of SP cells was clearly different from normal bipolar fibroblasts. They were stable and unaffected by cell densities in contrast to alterable morphology of normal fibroblasts. SP cells were n o t the normally accumulating aged cells. The SP cells induced by B [ a ] P may be the fibroblasts altered in their shape and n o t the selectively survived and increased SP-like cells already present in few numbers before B[a]P-treatment. This is suggested by the following: (a) The mass of SP cells appeared suddenly at 2 days after the B [ a ] P addition. The number of SP cells increased 52-fold from 1 to 2 days in 10 pM B[a] P-treated culture (Fig. 1). This 52-fold increase requires 6 cell divisions within 24 h, which would n o t normally occur. (b) Intermediate narrow cells were observed preceding the appearance of SP cells. (c) Arrangement of SP cells and their relation to epitherial colonies were almost the same with those of fibroblasts in control cultures. Fibroblasts are known to alter their morphology to elongated form in the presence of cAMP or other chemicals [7--11]. But, the induction of SP cells in MEC cultures by B [ a ] P is different from this. Alteration induced with cAMP or others occurs within a few hours after addition and is reversible when the inducing agent is removed from the culture medium [8,10]. On the other hand, induction of SP cells by B [ a ] P is delayed for 2 days after the addition of B[a] P. It was not reversible after the removal
47
of B[a] P. SP cells were maintained even after replating them to a fresh dish with fresh culture medium. Induction of SP cells in MEC by PAH is n o t a non-specific artifact. It did n o t occur in response to changes of pH of the medium as described by DiPaolo and Donovan [6]. SP cells did n o t appear spontaneously under abnormal or unsuitable culture conditions in temperature, osmolarity, or serum concentration. Suppression of cell growth and degradation of some cells were observed c o n c o m i t a n t l y with the appearance of SP cells. But these early cytotoxicities did n o t relate to the induction of SP cells, as both showed different dose-responses to B[a] P (Fig. 3). In various PAHs, only p o t e n t carcinogens (DMBA, MCA, B[a]P, and DB[a,e] P) induced SP cells in MEC cultures. Among these PAHs, those with the higher IbaU's indices [12,13] induced SP cells at lower dose and in a shorter time (Table II). Thus activities of PAHs to induce SP cells corresponded to the carcinogenicities of the chemicals, a N F is known as a p o t e n t inhibitor of arylhydrocarbon hydroxylase (AHH) [14]. As a N F suppressed the induction of SP cells by PAH, metabolism of PAH by AHH m a y be a prerequisite for the induction. This is further suggested by the fact t h a t when added after 24 h exposure to PAH, a N F did n o t prevent induction. DB[a,h] A, which has Iball's index o f 26, did n o t induce SP cells. It may be possible that the doses and/or its metabolic activation by the cells were insufficient for induction. Results obtained by 3-OH-B[a]P are interesting as 3-OH-B[a]P is a representative cytotoxic metabolite of B[a] P by AHH [15] which has no carcinogenic properties [16]. The main effects of 3-OH-B[a]P on HECs were regression and detachment of the cells, which were rapid and partially reversible, and n o t the induction of SP cells. Active metabolite(s) of B[a] P for the induction may be different from 3-OH-B[a] P. In conclusion, the appearance of SP cells in MEC cultures treated with PAH corresponds to the carcinogenicity of the chemicals. SP cells may be fibroblasts altered in their morphology irreversibly by the treatment with carcinogens. Appearance of SP cells may reflect some c o m m o n action of carcinogens on the cells and may be useful for rapid detection of carcinogens, though further studies are required. ACKNOWLEDGEMENT
I t h a n k Dr. N. Kinoshita (Medical and Clinical College, the Univ. of Kyushu) for providing 3-OH-B[a] P. REFERENCES
1 2 3 4
48
Y. Berwald and L. Sachs, J. Natl. Cancer Inst., 35 (1965) 641. J. Kamahora and T. Kakunaga, Biken J., 9 (1966) 295. T. Kuroki and H. Sato, J. Natl. Cancer Inst., 41 (1968) 53. N. Inui, S. Takayama and T. Sugimura, J. Natl. Cancer Inst., 48 (1972) 1409.
5 H. Tsuda, N. Inui and S. Takayama, Biochem. Biophys. Res. Commun., 55 (1973) 1117. 6 J.A. DiPaolo and P.J. Donovan, Exp. Cell Res., 48 (1967) 361. 7 R.P. Cox and B.M. Gesner, Proc. Natl. Acad. Sci. USA, 54 (1965) 1571. 8 G.S. Johnson, R.M. Friedman and I. Pastan, Proc. Natl. Acad. Sci. USA, 68 (1971) 425. 9 G.S. Johnson and I. Pastan, J. Natl. Cancer Inst., 47 (1971) 1357. 10 L. Diamond, S. O'Brien, C. Donaldson and Y. Shimizu, Int. J. Cancer, 13 (1974) 721. 11 G.S. Johnson and J.P. Schwartz, Exp. Cell Res., 97 (1976) 281. 12 J. Iball, Am. J. Cancer, 35 (1939) 188. 13 J.C. Arcos and M.F. Argus, Adv. Cancer Res., 11 (1968) 305. 14 L. Diamond and H.V. Gelboin, Science, 166 (1969) 1023. 15 H.V. Gelboin, E. Huberman and L. Sachs, Proc. Natl. Acad. Sci. USA, 64 (1969) 1188. 16 P.G. Wislocki, R.L. Chang, A.W. Wood, W. Levin, H. Yagi, O. Hernandez, H.D. Mah, P.M. Dansette, D.M. Jerina and A.H. Conney, Cancer Res., 37 (1977) 2608.
49