Oxygen and light effects on chromosomal aberrations in mouse cells in vitro

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Experimental Cell Research 104 (1977) 199-205

OXYGEN

AND LIGHT

ABERRATIONS

EFFECTS

IN MOUSE

ON CHROMOSOMAL CELLS

IN VITRO

R. PARSHAD,’ K. K. SANFORD,* G. M. JONES,* F. M. PRICE* and W. G. TAYLOR’ IDepartment of Pathology, Howard Vniversiv School of Medicine, Washington, DC 20059, and =LLaboratoryof Biochemistry, National Cancer Institute, National Institutes of Health, US Public Health Service, Department of Health, Education and Welfare, Bethesda, MD 20014, USA

SUMMARY Decreasing the oxygen concentration in the gas phase from 18% (atmospheric) to 1% decreased the frequency of chromosomal aberrations in mass cultures of cells from adult lung and embryos of two inbred mouse strains. Both the rate of shift from the diploid number and the incidence of abnormal chromosomes were decreased at the lower oxygen level. Similarly, shielding mouse cells from room lights (cool white, fluorescent) during routine fluid renewals reduced the incidence of abnormal chromosomes, particularly minutes and metacentrics. The increased incidence of chromosomal abnormalities on exposure of cells to light and high oxygen presumably results from a photodynamic reaction affecting the DNA or associated proteins of the chromatin fibers.

Because of the extensive genetic information on inbred mouse strains, mouse cells in culture are particularly useful for somatic cell hybridization and carcinogenesis studies. However, the chromosomes of mouse cells are unstable, and changes in number and structure may occur within the first 2 weeks in culture [l]. The type of serum used in the growth medium influences chromosome stability, since chromosomal abnormalities arise at a much lower rate in medium supplemented with fetal bovine serum (FBS) as compared with horse serum (HS) [2]. Data from another study [3] suggest a relationship between gaseous oxygen concentration and chromosome stability. Also, lowering the O2 con-

centration from atmospheric (18% v/v) to l-3 % v/v markedly enhances the plating efficiency of low passage, presumably euploid cells, from several species [4]. Near-UV and visible light in the presence of O2 can cause mutations in bacteria [5-8] and in the presence of riboflavin cause changes in the structure of DNA [9]. Light also produces toxic photoproducts in tissue culture medium in the presence of Oe [ 10-121. The objectives of these studies are twofold: (a) to determine if the use of a gas phase containing 1% O2 will promote chromosome stability in cultures derived from embryonic or adult mouse cells; (b) to ascertain whether exposure to light results in chromosomal aberrations. Exp Cell Res 104 (1977)

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wavelength (nm); ordinate: (left) rel. energy (%); (right) % transmission. Spectral distribution of light from Westinghouse F40 cool white fluorescent lamp and percent transmission of various wavelengths through O-O, plastic and O-O, Pyrex glass flasks. Three peaks in the photochemically reactive region of the spectrum occur at 430, 405 (40% of energy at 430) and 350 nm (19% of energy at 430) with 2 % of total energy below 375 nm. Note that Pyrex glass transmits more light than plastic which excludes all wavelengths below 295 nm.

Fig. 1. Abscissa:

MATERIALS

AND METHODS

Cells and culture procedures For the study of 0% effects cell line NCTC 8088 was initiated from a mince of 12-day C3H$HeN embryos and grown in NCTC 135 (supplemented with 0.5 PM ZnSO, .7 H,O) containing 10% stallion horse serum (HS) (Flow Laboratories, Rockville, Md). After the first subculture, the line was subdivided and cultured with a gas phase of 1% 0, : 10% CO*: 89% N2 (Paz in the medium 35-50 mmHg) or 18% O2: 10% CO, : 72% N, (PO2 in the medium 125-140 mmHg) (Air Products and Chemicals, Inc., Hyattsville, Md). Cell lines were also initiated from lung tissue of a 59-dayold C57B1/6N male mouse. This tissue was minced, washed in saline and treated with 0.25% trypsin (Difco 1 : 250). Dispersed cells were grown in NCTC 135 supplemented with 10% HS and carried at 1% Oz (NCTC 8468) and at 18% 0, (NCTC 8469) or in 135 supplemented with 10% fetal bovine serum (FBS) (Flow Laboratories) and carried at 1% Oz (NCTC 8466) and at 18% O2 (NCTC 8467). For the first 2 weeks 50 &ml gentamicin (Schering Corp., Kenilworth, N.J.) was added to the culture medium. Subsequently, cultures were tested and found free of mycoplasma and other microbial contaminants. For the study of light effects, three pairs of cell lines were initiated each from a mince of 11-IZdayold C3HI/HeN embryos. One member of each pair (NCTC 8505, 8507, 8547) was carried in T-15 flasks wrapped in aluminum foil to shield the cells from light. The other member of the pair (NCTC 8504, 8506, 8546) was carried in T-15 flasks exposed to the usual lighting conditions. These conditions included -3O-min exposure of cultures and l-3-h exposure of medium to room lights three times weekly for medium renewals and subculturing and variable short exExp Cell Res 104 (1977)

posures during incubation in a walk-in incubator. The room lights (Westinghouse F40 cool white) had an intensity of -100 foot candles at the bench top. The spectral distribution of the lamps is presented (fig. 1) with the percent transmission through both plastic and Pyrex glass of culture flasks. The shielded cultures were exposed to light for a few seconds once a week when examined on an inverted microscope equipped with a tunnsten filament lamo shielded with daylight and heat iTtiTters.Cells wera &own in Dulbecco Vogt’s (DV) modified MEM supplemented with 10% FBS as described above and were gassed with 10% CO* in air (18% 02). After 9 and 15 days one culture of each line was transferred to and serially subcultured in DV supolemented with 10% HS (stallion). The cell lines on HS were designated NCTC 8520 (from 8505). 8522 (from 8507), 8547A (from 8547), 8519 (from 8504) 8521 (from 8506) and 8546A (from 8546). Cells were grown in 3 ml medium which was renewed three times a week; confluent T-15 cultures were split 1: 2 by mopping cells from the growth surface with perforated cellophane [ 131except during the first five transplant generations of the adult lung cells which were dispersed with trypsin. No antibiotics were used except as noted above. Cultures were gassed at each fluid renewal with a humidified gas mixture as described above.

Chromosome preparation analysis

and

Each line was examined for chromosomal alterations after variable periods in vitro by procedures described 1141; 100 intact metaphase plates were selected at random and examined for chromosome number and structural alterations.

RESULTS Oxygen effect Mouse embryo cells with 1% 0, in the gas phase, as compared with 18% 02, maintained a higher frequency of diploid (2n =40) (46 % vs 32 %) and tetraploid cells (28 % vs 12%) during the first 35 days in culture. Thereafter, both lines became heteroploid with chromosome numbers predominantly in the subtetraploid range. However, the cells exposed to 1% 0, had a consistently lower number of abnormal chromosomes (table 1, line 8088). Cultures of adult mouse lung cells gassed with 1% O2as compared with 18% O2maintained a significantly higher frequency of diploid cells during the entire period of

Oxygen and light effects on chromosomal 1%0,

10%o*

I

I

II.

/

I

aberrations

201

by the rate at which the cultures could be split 1: 2 when confluent. In BBS-supplemented medium, cells grown with a gas phase of 1% O2 exhibited no abnormal chromosomes until 202 days in vitro when two metacentrics were observed in a sample of 100 metaphase cells (table 1, line 8466, 8467). By comparison, cells grown at 18% O2 exhibited abnormal chromosomes as early as 56 days and in all subsequent analyses, ranging from 5 to 32 100 metaphase cells. In HS-supplemented medium, cells grown with a gas phase of 1% 0, exhibited a maximum of 14 minutes (defined as less than half the length of the shortest chromosome in the normal mouse karyotype) and two metacentrics as compared with 40 minutes and 16 metacentricsl 100 metaphase cells in cultures at 18% O2 (table 1, lines 8468, 8469). Consistent differences between the two experimental

Fig. 2. Abscissa: no. of chromosomes/cell; ordinate: % of cells analysed. Effect of OI concentration in the gas phase on distribution of chromosome numbers in C57B116N adult lung cells grown in NCTC 135+10% fetal bovine serum.

study (figs 2, 3). Even after 202 days in vitro cultures gassed with 1% 0, contained 74% and 18% diploid cells in BBS and HS supplements, respectively. On the other hand, when gassed with 18% O2 cultures contained only 38 % and 0 % diploid cells in BBS and HS supplements, respectively. The higher number of diploid cells at 1% 0, could not be attributed to a lower proliferation rate; cells grown at 1% O2 proliferated as rapidly as those at 18% 02, as estimated

Fig. 3. Abscissa: no. of chromosomes/cell; ordinate: % of cells analysed. Effect of O2 concentration in the gas phase on distribution of chromosome numbers in C57B1/6N adult lung cells grown in NCTC 135+ 10% horse serum. Exp Cell Res 104 (1977)

202

Parshad et al.

Table 1. Efiect of O2 concentration on frequency of abnormal chromosomes in mouse cell lines grown in NCTC 135 supplemented with horse serum (HS) orfetal bovine serum (FBS) Gaseous 0, concentration

NCTC cell line

Serum supplement

8088

HS

8468,8469

8466,8467

HS

FBS

Days in vitro 35 126 154 56 125 158 202 56 109 125 158 202

18%

1%

Metacentrics Other” nc Minutes EC

Metacentrics Other” nc Minutes nc

A

20

NDd 4

2 10 ND 1

ND 1

4 0

40 12 40 5 8 8 28 12

86 16 0 2 2 4 4

8 0 0 2 2 0 0

: 14 8

3;

0 0 0

Pb

0 1 2 0

0 0 0

0.002
0

:, 2 0 0 0 0 2

8 0

0.014 0.0004
0 0 0 0 0

a Other, chromatid break or Sap, heterozygous translocation. b Probabilities based on combining the three types of abnormalities. c R, number per 100cells. d ND, not determined.

groups were observed in all analyses. Representative metaphase fgures of cells of these lines are presented (fig. 4). Light effect Light exposure did not substantially affect the distribution of chromosomes in the three pairs of lines grown for 29-3 1 days on FBS-supplemented medium. The percentage of diploid cells in the unshielded lines was 54 %, 74 % and 78 % as compared with 57%, 68 % and 82 % in the shielded. When unshielded cultures of all three lines were transferred to HS, the incidence of diploid cells decreased to 38%, 23 % and 58 % respectively; however, when shielded cultures were transferred to HS little or no change in distribution of chromosome numbers occurred and 60%, 56 % and 68 % respectively of the cells remained diploid. Light exposure increased the frequency of abnormal chromosomes in one of the Exp Cell Res 104 (1977)

three lines grown in FBS-supplemented medium and in all three lines transferred to HS-supplemented medium (table 2). An effect of light was thus clearly demonstrated especially when cells were grown in HSsupplemented medium. Proliferation rates of all cell lines were similar as estimated by the rate at which cultures could be split 1: 2 when confluent. DISCUSSION These results indicate that molecular oxygen and visible light produce chromosomal aberrations in mouse cells in vitro. Decreasing oxygen concentration in the gas phase from atmospheric (18 %) to 1% decreased the incidence of abnormal chromosomes and the rate of shift from the diploid number during long-term culture. Similarly, shielding cultures from room lights decreased the incidence of abnormal chromo-

Oxygen and light effects on chromosomal aberrations

203

Fig. 4. Representative metaphase figures from lines of C57Rl adult lung cells (NCTC g466-Wj9) after 125 days in culture grown in medium NCTC 135 supplemented with (A, B) 10% FRS or (C, D) 10% HS. Cells in (A) and (C) were subjected to 1% 0, and (8, D) to 18% OS. Note diploid karyotype in (A) and (C) with 40 chromosomes, one chromosome in (A) having a

secondary constriction; in @) 38 chromosomes with a large metacentric chromosome and in (D), a subtetraploid number with one large metacentric (a), two minute chromosomes (b), and a chromosome with chromatid break/gap (c). (A) x748; (B) x872; (C) x 1050; (0) x 1 140.

somes even at atmospheric oxygen concentrations. The abnormal chromosomes were predominantly minutes and metacentrics. The minute chromosomes result from breaks in chromatin fibers during

interphase with loss of acentric fragments from the fibers. Metacentric chromosomes result from breaks in two chromatin fibers and subsequent rejoining of the broken ends of the acentric fragment of one with the Exp Cell Res 104 (1977)

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Parshad et al.

Table 2. Influence of shielding cells from fluorescent room lights on the frequency of abnormal chromosomes in cell lines grown in Dulbecco-Vogt’s medium supplemented with fetal bovine serum (FBS) or horse serum (HS) Shielded

Unshielded NCTC cell line

Serum supplement

8504,8505

FESS

8506,8507 8546,8547 8519,852O 8521,8522 8546A, 8547A

HS

Days in vitro

Metacentrics Minutes nc

31 71 31 29 31 31 29

11 16 4 2 30 33 10

3 4 0 0 4 13 6

Other” nc 1 i 0 0 8

Metacentrics Other” Minutes tre nc

P*

2 2 2 0 6 4 8

0.001 <0.0001 0.34 0.24
0 0 0 0 0 10 0

0 1 0 0 0 2 0

a Other, chromatid break or gap; heterozygous translocation. * Probabilities based on combinina the three tvnes . . of abnormalities. ’ n, number per 100 cells.

centric fragment of the other [IS]. In view of these origins of the abnormal chromosomes, we conclude that light and high oxygen concentration increase the frequency of chromatin breaks. The near-UV and visible light to which the medium and cells were exposed during routine handling in the present study have been shown to affect both bacteria and mammalian cells in a variety of ways. In bacteria, near-UV has been shown to be mutagenic [5-71 and to produce O,-dependent damage. Much of the damage produced by light of 365 nm wavelength has been identified as excision-reparable lesions in DNA [8]. Since DNA does not measurably absorb at 365 nm, the DNA damage presumably results from absorption of energy by a cellular component other than DNA followed by a photo-oxidation of DNA. In mammalian cells, growth-inhibitory or lethal effects have been produced by exposing the culture medium to near-UV and visible light [ 10-121. The cell killing resulted from the formation of toxic photoproducts from riboflavin and tryptophan (or tyrosine) EXF Cell Res 11% (1977)

in the medium. The reaction occurred only at low cell density and was oxygen-dependent [ 121.Photoactivated riboflavin has also been shown to produce extensive changes in the structure of purified calf thymus DNA. These changes are thought to result from a primary photochemical effect on the guanine moiety. Limited data on HeLa cells suggested that a photochemical reaction also occurs in living human cells in culture as evidenced by the lowered molecular weight of DNA from cells illuminated in the presence of large amounts of riboflavin [9]. From known effects of light and oxygen in photosensitizing riboflavin and other chromophores to produce oxidative changes in amino acids, proteins, and DNA [16-181, we postulate that the increased frequency of chromatin breaks observed in the present study results from a photodynamic reaction affecting the DNA or associated proteins of the chromatin fiber. Further, the production of genetic alterations by exposure of cultures to light and atmospheric oxygen during routine handling may be the causative factor in spontaneous neoplastic transformation [ 191.

Oxygen and light effects on chromosomal We are grateful to Dr Robert E. Tarone of the Biometry Branch, NCI, for the statistical analysis of data.

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10. Warburg, 0, Geissler, A W & Lorenz, S, Z klin Chem klin Biochem 6 (1968) 467. 11. Freeman, R G, Arch dermatol 102 (1970) 521. 12. Wang, R J, In vitro 12 (1976) 19. 13. Evans, V J, Bryant, J C, Kerr, HA & Schilling, E L, Exp cell res 36 (1964) 439. 14. Taylor, W G & Parshad, R, Methods in cell biology (ed D C Prescott) vol. 15. Academic Press, New York (1976). 15. Parshad, R.& Sanford, K K, J natl cancer inst 41 (1968) 767. 16. Spikes, J D & MacKnight, M L, Ann NY acad sci 171 (1970) 149. 17. Knowles, A, Res prog erg biol med them 3 (1972) 183. 18. Santamaria, L & Prino, G, Res prog erg biol med them 3 (1972) XI. 19. S$ord; K K, J natl cancer inst monog 26 (1%7)

Received March 10, 1976 Accepted August 19, 1976

Exp Cell Res 104 (1977)