The effect of tissue culture agar on chromosome breakage, sister-chromatid exchanges and clonogenicity in human cells

Mutation Research, 208 (1988) 201-205

2(11

Elsevier MTR 01291

The effect of tissue culture agar on chromosome breakage, sister-chromatid exchanges and clonogenicity in human cells M . M . C o h e n 1,2, S. S c h w a r t z l, A . K u n s k a

1, j . S a t i s h i a n d A. H a m b u r g e r

3

I Division of Human Genetics, Departments of Obstetrics/GvnecoloKv and Pediatrws, Unwerst O' of Mau,'land at Bahtmore Medical School, : Medical Biotechnology Center of the Maryland Biotechnologv Institute. Baltimore, and ~ UnwersiO, of Maryland Cancer ('enter, UniversiO, of Ma~. 'land at Bahimore Medical School, Balttmore, MD ( U S . A )

(Received 31 August 1987) (Revision received 19 November 1987) (Accepted 21 l)ecember 1987)

Keywords." Agar, tissue culture; Chromosome breakage, effect of tissue culture agar; Sister-chromatid exchanges, effect of tissue

culture agar; Human cells, effect of tissue culture agar; Clonogenicity, effect of tissue culture agar Summa~' To investigate the cytogenetic effects of electromagnetic fields, a system containing an agar gel was dew:loped to support the growth of various human cell types (peripheral lymphocytes, lymphoblasts, and fibroblasts). When compared to alioquots of identical cells, grown in plastic culture vessels, statistically significant increases in the frequencies of chromosome breakage, sister-chromatid exchange and decreased cloning efficiency were observed in those cells cultured in the agar. These results suggest a possible clas~ogenic a n d / o r eytotoxic component in the agar gel.

Recently, we reported the effects of extremely low-level electromagnetic fields (EMF) on several cellular parameters in a variety of human cell types. The endpoints evaluated included cell cycle duration, mitotic rate, frequencies of chromosome breakage and sister-chromatid exchange (SCE), and clonogenicity in soft agar (Cohen et al., 1986a, b). The apparatus used a support gel consisting primarily of tissue culture agar, containing wells in which the cells were suspected. This communicatic, n reports observations comparing the above endpoints in non-exposed control cells which had

Correspondence: Dr. M.M. Cohen, Division of Human Genetics, Departments of Obstetrics/Gynecology and Pediatrics, University of Ma~land at Baltimore Medical School, Baltimore. MI) (U.S.A.).

been placed in the agar gel with those cultured in the standard tissue culture vessels. Materials and methods

The system designed for the delivery of uniform electrical and magnetic fields of specified intensities has been described in detail elsewhere (Cohen et al., 1986a, b). The e x p o s u r e vessel. A polypropylene sterilizing tray measuring 8.6 cm x 17 cm x 5 cm (Fisher Scientific) was filled to a depth of 4.5 cm with a sterile 1.5% agar gel made as follows: 10.5 g of tissue culture agar (Difco) dissolved in 350 ml of Hanks' balanced salt solution (Gibco), and 3.5 ml N a e H C O 2 (Gibco). The liquid was autoclaved and then poured into the sterile tray and mixed

0165-"992/88/$03.50 '3:1988 Elsevier Science Publishers B.V. (Biomedical Division)

202 with 350 ml of RPMI 1640 (Gibco), containing Hepes buffer for maintenance of pH, in an approximate 1 : 1 ratio (total volume of 700 ml). The solution was allowed to cool and before "gelling", 4 symmetrical tapered 5-ml wells, cast on 4 c m x 4 cm centers, were made using a preformed sterile Teflon template (Cohen et al., 1986a, b). The exposure vessel was placed in a specially modified Napco, Inc. Series 6100 tissue incubator (37.5 °, 5% CO 2 in air) for incubation,

Cells and cell culture Peripheral blood lymphocvtes. A group of 10 healthy, normal individuals (5 males and 5 females) was used throughout these studies. Heparinized peripheral blood samples were obtained, and 0.25 ml of whole blood was added to 5.0 ml of the identical RPMI 1640 tissue culture medium containing 20% fetal calf serum, phytohemagglutinin, and antibiotics, and placed in each well of the gel. Individuals from this group were utilized in pairs (one of each sex), and samples were replicated in 2 of the 4 wells in the gel. Identical cultures were established in regular flasks or tubes and placed in the incubator. At the end of the incubation period (69 h), colcemid (0.02 ffg/ml) was added to each well a n d / o r flask (tube) for 1 h to accumulate metaphases. The cells were harvested according to a modification of a standard procedure and nonbanded, Giemsa-stained chromosome preparations were made (Moorhead et al., 1960). Lymphoblastoid cell lines (LCL, s'). Cells from 5 LCLs were studied: one normal individual ( G M 892); and one patient each with ataxia telangiectasia (AT) (GM 1525); xeroderma pigmentosum (XP) (D 7031); Bloom syndrome (BL) (GM 4408); and Fanconi anemia (FA) (HSC 230). All lines with G M designations were obtained from the H u m a n Genetic Mutant Cell Repository, Camden, N J; the XP and FA lines were gifts from Dr. Bernard Strauss (University of Chicago) and Dr. Manuel Buchwald (Hospital for Sick Children, Toronto, Ontario, Canada). All cells, cultured in medium RPMI 1640 supplemented with Hepes buffer containing 20% heat-inactivated fetal calf serum, antibiotics, and additional L-glutamine, were maintained in culture

flasks at 37 o C in an atmosphere of 5% CO2 in air. The cultures were "split" when the cell concentration reached 1 - 2 x 106 cells/ml. Subculturing was accomplished by dividing the culture into several flasks and adding fresh medium to achieve the proper dilution. Cells of each line were seeded into the wells of the exposure gel at a final concentration of 5 x 104/ml for 69 h and harvested as described above for lymphocytes.

Human cancer lines. Two well-characterized human colon carcinoma cell lines (Colo 205 and Colo 320DM) were obtained from the American Type Culture Collection, Rockville, MD. Colo 205 (CCL 222) was directly derived from the ascitic fluid of a patient with adenocarcinoma of the colon (Semple et al., 1978), while the Colo 320DM (CCL 220) was derived from an undifferentiated adenocarcinoma of the sigmoid colon (Quinn et al., 1979). Both lines were passaged as monolayer cultures in RPMI 1640 medium containing 10% fetal bovine serum (FBS), Hepes buffer, and antibiotics. Preliminary experiments established the growth kinetics of these lines to assure that all cells used experimentally were in the logarithmic growth phase and within 15 passages of the frozen stock. Viability counts by the trypan blue exclusion method yielded values consistently greater than 90%. Parameters studied Chromosome damage. For evaluation of chromosome breakage in peripheral lymphocytes and each ECL, two microscope slides were prepared from the cells in each well in the agar gel or culture tube, numerically coded, and analyzed "blindly" without knowledge of the identify or treatment. Chromosome damage was quantitated as previously described by Cohen et ai. (1983). 50 metaphases per replicate (100 metaphases/pers o n / t r e a t m e n t ) were analyzed in all cases. Structural rearrangements, including dicentric chromosomes, multiradial configurations, rings, and obvious translocations were considered as two break events; chromatid and isochromatid breaks were scored as single breaks. Chromosome breaks were totalled and reported as the mean number of breaks per cell.

203

Sister-chromatid exchanges. Cells from both peripheral lymphocytes and LCLs were introduced into culture as described above. 5-Bromodeoxyuridine (BrdU) (Sigma) was added at a final concentration of 10 p.g/ml for the last 48 h of incubation, during which time the incubator remained closed to minimize SCE induction by photolysis of BrdU-substituted D N A . Colcemid (0.02 p.g/ml) was added for the final 60 rain of culture. Harvesting of cells and slide preparation were accomplished by the FPG method of Perry and Wolff (1974). The slides were stained for 12 min in a 0.5 /~g/ml solution of the fluorochrome Hoechst 33258, exposed to a mixture of both longand short-wave UV light (distance approximately 8 in.) for a minimum 15 rain at 5 5 ° C in McIlvaine's buffer (pH 8.0), rinsed, and stained with 3% Harleco Giemsa at pH 6.8. The microscope slides were code-labeled with numbers only and exantined "blindly", without knowledge of source. 20 sequentially selected second-division meta-

phases (lO/culture well) per individual or line were studied, and the number of S C E / c e l l recorded.

ClonogeniciO, assays. The soft agar cloning method of Hamburger and Salmon (1977a,b) was used in these studies. A base layer of 1 ml of 0.5% agar (Difco) containing RPMI 1640 medium and 10% FBS was prepared in 35-mm plastic petri dishes. A 1-ml overlay of 0.3% agar containing 5 x 103 Colo 205 or Colo 320DM cells in RPMI 1640 was applied over the agar base. Plates were incubated at 3 7 ° C in a humidified atmosphere of 5% CO: in air, and colonies consisting of more than 40 cells were counted 7 days after plating using an inverted phase microscope or a Bausch and Lomb FAS II image analyzer.

Statistical analysis. Since all specimens were tested in pairs (i.e., agar gel vs. culture flask), each person, LCL, or cell line served as its own control.

TABLE 1 R E S L L T S O F V A R I O U S C Y T O L O G I C A L P A R A M E T E R S ( m e a n -+ S.D.) O B S E R V F . D IN C E L L T Y P E S C U L T U R E D A G A R G E l . A S C O M P A R E D T O P R O P A G A T I O N IN R E G U L A R T I S S U E C U L T U R E V E S S E L S

Cell t3pe

Chromosome breaks/cell Agar gel

Cuhure flask

Paired value

P value

t

Peripheral b l o o d ( N ,= 10 i n d i v i d u a l s ) L('L Normal BL AT XP FA

(N (N (N (N (N

= = = = =

3 3 3 3 3

trials) trials) trials) trials)

trials)

0.034 + 0.024

0.001 _+0.0003

0.317 -+ 0.035 0.127+0.025 0.180+0.043 0.090-+0.010 0.553 + 1.480

0.033 + 0.023 0.007 -+ 0.015 0.030-+0.010 0 . 0 1 7 + 0.015 0 . 1 2 0 + 0.020

4.26

0.002

12.14 5.76 7.50 5.50 7.60

0.007 0.029 0.017 0.032 0.017

< 0.001

Sister-chromatid exchanges/cell Peripheral bkK~d ( N =: 10 individuals)

17.890_+3.22

8.09+_ 1.18

10.32

13.95 14.32 16.67 11.52

6.76±0.65 4.54+_0.83 3.51 _+ 1.13 6.63 + 0.71

7.17 14.33 43.58 4.95

I.('L

Normal ( N = 3 trials) BL ( N = 3 trials) AT ( N = 3 trials) XP ( N = 3 trials)

_+1.18 __+0.65 _+0.65 _+ 1.00

0.019 O.OO5 0.001 0.038

Clonogenicity assay (number of colonies) l.ine ( ' o h ) 205 Colo 320DM

( N = 9 trials) ( N = 9 trials)

520.0 + 100.8 7 2 5 . 6 + 66.1

1 197 + 275.4 1 141 + 161.7

2.88 2.88

0.021 0.021

1N " I f t E

2o4 This experimental design is best suited for analysis by a paired t test. The level of statistical significance for all comparisons was pre-established as P < 0.05. Results

Table 1 describes results obtained from cells cultured in the control incubators for the 3 parameters studied: chromosome breakage, SCE frequency, and cloning ability in soft agar. In general, those cells which had been grown in the wells of the agar gel demonstrated significantly greater "adverse" effects than those cultured in regular plastic vessels. These were not isolated findings nor single evaluations, but were based on repeated trials for each cell type. With regard to the peripheral lymphocytes, for each of the 10 panel members, a regular culture tube was included with his or her gel sample on at least 3-5 occasions; each LCL was tested 3 times and the two cancer cell lines were repeated 9 times each. The consistent nature of the observation is evidenced by the narrow standard deviations associated with each parameter during multiple tests over an extended time period. The chromosomal aberrations observed were widely distributed among the cell population studied, and almost invariably were limited to 1 or 2 per cell. They do not represent cells with extensive damage (multibreak cells) which may be rare outliers and skew the data. The data presented in Table 1 show statistically significant differences for every comparison based on the paired t test results. It should also be pointed out that these cells (both from individuals of the peripheral lymphocyte panel and the LCLs) have been routinely cultured in regular vessels for other types of experiments. Control values (i.e., culture flasks) for chromosome damage and SCEs from the present experiments were compared to flask results obtained from other studies using the same cells in different incubators and laboratories showed no significant differences (data not shown). For each comparison, cells which has been in contact with the agar gel demonstrated significant increases in both chromosome damage and SCE frequency. Similarly, decreased cloning efficiency was noted for both the Colo 205 and Colo 320DM

cell lines grown in the agar gel. These observations strongly implicate the agar gel, or some ingredient thereof, as having possible clastogenic and cytotoxic potential.

Discussion

Cellular exposure to physical a n d / o r chemical environmental agents classified as putative clastogens, mutagens, teratogens, or carcinogens is frequently demonstrated by increased levels of spontaneous chromosome damage and sister-chromosome exchange. Many biologically active agents can significantly increase chromosome breakage which in somatic cells may be associated with a propensity for neoplastic development, while in germ cells may lead to a higher frequency of spontaneous abortions, birth defects, and heritable chromosomal rearrangements (Hsu, 1983). Sister-chromatid exchange may represent normal repair of D N A damage and therefore has been used extensively to assess the DNA-damaging potential of various physical and chemical environmental agents ( L a t t e t al., 1980; Tice et al., 1984). SCEs are more easily induced by chemical rather than physical agents (Natarajan et ai., 1980; Evans and Vijayalaxmi, 1981; Fornace et al., 1980; Perry and Evans, 1975; Popsecu et al., 1977: t+att and Schreck, 1980), and apparently result from a mechanism independent of chromosomal breakage (Wolff, 1978; Lin and Wertelecki, 1982; Hedner ct al., 1982). It was surprising to observe significant increases in both SCE and chromosome breakage frequencies in cells grown in the agar gel when compared to the plastic culture vessels normally used for their propagation. A similar detrimental effect on cloning efficiency was recorded in the colon cancer cells grown in the agar wells. That these results are meaningful is indicated by the fact that the phenomenon was consistent and repeatable, being noted in replicated experiments utilizing 3 different cell types (PHA-stimulated peripheral leukocytes, EB virus-transformed B cells, and cultured fibroblasts). These observations were made over a 4-year period of time (1/83-12/86), during which many different batches of tissue culture agar were used. There-

205 fore, the results c o u l d not be a t t r i b u t e d to a single b a t c h or lot of agar. T h e p r o b l e m o f a t t e m p t i n g to d e t e r m i n e w h i c h c o m p o n e n t o f the a g a r m a y be r e s p o n s i b l e for the o b s e r v a t i o n s is v e r y difficult. B e i n g d e r i v e d f r o m a natural product introduces a significant amount of v a r i a b i l i t y d u e to the b i o l o g i c a l n a t u r e o f the s o u r c e material. F u r t h e r c o m p l i c a t i o n s m a y b e i n h e r e n t in the p r e p a r a t i o n o f the gel for the e x p e r i m e n t s p e r f o r m e d . T h a t is. the gel was m i x e d w i t h tissue c u l t u r e m e d i u m , a n d a u t o c l a v e d a n d p l a c e d in the sterile plastic c o n t a i n e r s w h i l e still v e r y hot. T h e o b s e r v e d effects m i g h t b e a t t r i b u t a ble to an i n t e r a c t i o n o f these m a t e r i a l s in the fornaation o f a c o m p o n e n t i n d u c i n g the i n c r e a s e d c h r o m o s o m e d a m a g e a n d S C E f r e q u e n c y a n d red u c e d c l o n o g e n i c i t y . O n l y f u r t h e r studies o n individLal i n g r e d i e n t s of the agar, a l o n e an in c o m b i nation, and comparative biochemical analyses of m e d i u m f r o m the a g a r wells a n d tissue c u l t u r e vessels m i g h t e l u c i d a t e the p u t a t i v e c l a s t o g e n .

Acknowledgement T h i s s t u d y was p a r t o f a r e s e a r c h p r o g r a m to d e t e r m i n e the p o s s i b l e a d v e r s e h e a l t h e f f e c t s res u i t i n g f r o m e x p o s u r e to the electric a n d m a g n e t i c fields of o v e r h e a d h i g h - v o l t a g e t r a n s m i s s i o n lines. T h e p r o g r a m was a d m i n i s t e r e d by the S t a t e o f N e w Y o r k D e p a r t m e n t of H e a l t h R e s e a r c h , Inc. ( C o n t r a c t s N o . 212-082-11 a n d 8132182C). T h e views e x p r e s s e d h e r e i n a n d t h o s e o f the a u t h o r s a n d are n o t to be c o n s t r u e d as r e f l e c t i n g the p o s i t i o n o f the D e p a r t m e n t of H e a l t h o r H R I .

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