343
Mutation Research, 54 ( 1 9 7 8 ) 3 4 3 - - 3 5 4 © E l s e v i e r / N o r t h - H o l l a n d B i o m e d i c a l Press
SISTER-CHROMATID EXCHANGE INDUCTION BY SODIUM SELENITE: DEPENDENCE ON THE PRESENCE OF RED BLOOD CELLS OR RED BLOOD CELL LYSATE *
J A M E S H. R A Y a n d L E W I S C. A L T E N B U R G
University o f Texas Health Science Center, Graduate School o f Biomedical Sciences, Medical Genetics Center, P.O. Box 20334, Astrodome Station, Houston, Texas 77025 (U.S.A.) ( R e c e i v e d 22 M a y 1 9 7 8 ) ( R e v i s i o n r e c e i v e d 11 A u g u s t 1 9 7 8 ) ( A c c e p t e d 22 A u g u s t 1 9 7 8 )
Summary Sodium selenite (Na2SeO3) sister-chromatid exchange (SCE) induction was studied in both short-term and long-term cell cultures. The ability of Na2SeO3 to induce SCEs was found to depend on the culture conditions employed. Concentrations of Na2SeO3 (7.90 × 10 -6 M and greater) that produced elevated SCE frequencies in whole blood cultures resulted in control level SCE frequencies (6--8 SCEs/cell) in Ficoll--Hypaque -- purified lymphocyte cultures. However, whole blood and purified lymphocyte cultures were equally sensitive to SCE induction by methyl methanesulfonate (MMS), ethyl methanesulfonate (EMS), and N-hydroxy-2-acetylaminofluorene (N-OH-AAF). Analysis of different whole blood components showed that the presence of red blood cells (RBCs), and specifically RBC lysate, was a prerequisite for Na2SeO3 SCE induction in purified lymphocyte cultures. The SCE frequencies of xeroderma pigmentosum (XP12RO) and normal human lymphoblastoid cell lines were also found to be unaffected by Na2SeO3 concentrations that produced elevated SCE frequencies in whole blood cultures. Incubation of these latter two cell types with Na2SeO3 and RBC lysate resulted in SCE frequencies comparable to those in Na2SeO3-exposed whole blood cultures.
* A preliminary report of this work was presented at the 9th annual m e e t i n g o f t h e Environmental M u t a g e n Society, March 9--13, 1978, San Francisco, CA (U.S.A.).
Abbreviations: BrdU, bromodeoxyuridine~ EMS, ethyl methanesulfonate; FCS, fetal calf serum~ FdU, fhiorodeoxyuridine; HBSS, Hanks balanced salt solution; His +. histidine; MEM, minimum essential medium~ MMS, methyl methanesulfonate: Na2EDTA , disodium etbylenediamine tetraacetate~ N-OH-AAF, Nohydroxy-2-acetylaminofluorene~ RBC(s), red b l o o d cell(s)~ SCE(s), sisterchromatid exchange(s); U, uridine;/~ +- S.E., mean -+ standard error.
344 Introduction Sister-chromatid exchange (SCE) induction [ 1,6,20--22,34,39 ], unscheduled thymidine incorporation [24,35--37], and His + revertant production [18] all serve as sensitive indicators of DNA damage resulting from the exposure of mammalian and bacterial cells to chemical carcinogens and mutagens. A compound that exhibits the first two of these characteristics [17,23] is sodium selenite (Na2SeO~). Selenium-induced tumors have also been reported, but the data were criticized for the lack of proper controls [25,32 for reviews]; consequently, Na2SeO3 is not presently considered to be a carcinogen. In fact it exhibits anticarcinogenic [4,7,9,26--29,31], antimutagenic [10], and anticlastogenic [30] properties when used in combination with carcinogens, mutagens, and clastogens. Yet when Na2SeO3 is used alone, it is clastogenic [19], induces SCEs [23], and causes a significant amount of unscheduled DNA synthesis following incubation with crude liver extract [17 ]. Incubation with crude liver extract results in the activation of a group of compounds known as procarcinogens. These compounds generally do not induce SCEs [20,34,38], do not cause unscheduled DNA synthesis [24,35-37], and are not mutagenic [18] in in vitro tests because the organisms and cell cultures are not capable of converting them to proximate and ultimate carcinogenic forms. Following in vitro incubation with crude liver extract [2 ], however, these compounds exhibit the characteristic properties of carcinogens and mutagens. Thus, the available evidence suggests that while Na2SeO3 is on the one hand an anticarcinogenic/antimutagenic/anticlastogenic agent, it may also be a procarcinogen which can be activated under suitable conditions. In view of the conflicting results that have been obtained as to the carcinogenic potential of Na2SeO3 further study of Na2SeO3 SCE induction appeared warranted. The results of the experiments reported here demonstrate that Na2SeO3 alone does not induce SCEs when added to most culture systems. However, co-incubation of Na2SeO3 with red blood cells or red blood cell lysate results in its "activation" so that elevated SCE frequencies are produced. Materials and methods
Culture conditions Whole blood fractionation. L y m p h o c y t e s were purified from whole blood by the Ficoll--Hypaque gradient separation technique of B o y u m [3] with certain modifications. Whole heparinized blood that had been diluted with an equal volume of sterile 0.9% NaC1 was layered onto Ficoll--Hypaque gradients consisting of 4 parts 8% Ficoll (Pharmacia) and 1 part 50% Hypaque (Winthrop Labs). The ratio of diluted blood to gradient was 4 : 1. After centrifugation (400 × g; 40 min; 25°C) the lymphocytes, which appeared as a cloudy ring at the interface between diluted blood and the Ficoll--Hypaque gradient, were removed with a sterile Pasteur pipette and placed in tubes partially filled with Hanks Balanced Salt Solution (HBSS) (GIBCO). The tubes were then totally filled with HBSS, and the l y m p h o c y t e s were pelleted b y centrifugation (900 r.p.m.; 15 min; 25°C). Following supernatant removal and l y m p h o c y t e resus-
345 pension in HBSS, the centrifugation step was repeated. The final l y m p h o c y t e pellet was resuspended in Chromosome Medium 1A (GIBCO). Several whole blood components were prepared for addition to the cell cultures. Plasma from heparinized blood was obtained b y removing the supernatant of centrifuged whole blood. For collection of serum from coagulated blood, unheparinized whole blood was left at room temperature for at least 3 h. The upper liquid layer was then centrifuged (675 Xg; 5 min) and the supernatant fraction removed. Red blood cell (RBC) samples were obtained from the RBC pellets of Ficoll--Hypaque purified lymphocytes. The pellets were washed twice with Minimum Essential Medium (MEM) (GIBCO) and resuspended in Chromosome Medium 1A to a final volume equal to the original blood volume. For RBC lysis a volume of sterile, deionized water (4°C) equal to the packed volume of RBCs from 30 ml of whole blood was added to a centrifuge tube, and the suspension was agitated with a glass rod for 2 min. Following centrifugation (10 000 Xg; 10 min; 4°C) the RBC lysate was dispensed in 1.0-ml aliquots and stored frozen until use. The pellet containing RBC ghosts was repeatedly washed with 30-ml quantities of MEM and resuspended in water until microscopic examination revealed an absence of intact cells. The final RBC ghost pellet was resuspended in 30 ml Chromosome Medium 1A and stored frozen in 1.0-ml aliquots. The RBC lysate and ghosts were thawed immediately before addition to the cultures. Blood cultures. Whole blood cultures were initiated b y adding 0.3 ml of whole heparinized blood to 4 ml Chromosome Medium 1A. L y m p h o c y t e cultures were prepared by adding approximately 1 X 106 purified lymphocytes to 4 ml Chromosome Medium 1A. Three healthy individuals served as blood donors. Individuals I and III were males; individual II was female. Purified l y m p h o c y t e and whole blood cultures were incubated at 37°C for 96 h. Whole RBCs (0.3 ml) were added, when desired, to the purified lymp h o c y t e cultures at the beginning of the incubation period. After 53 h of incubation 1 X 10 -s M bromodeoxyuridine (BrdU), 4 X 1 0 -7 M fluorodeoxyuridine (FdU), and 6 X 1 0 - 6 M uridine(U) were added to the cultures [14]. BrdU served as both a thymidine analogue and an inhibitor of ribonucleotide reductase while F d U and U were added to inhibit thymidylate synthetase activity and to offset possible adverse effects of FdU on R N A synthesis, respectively [11]. After another 24 h the cells were pelleted (675 × g; 5 min), washed with 4 ml MEM, and resuspended in 4 ml Chromosome Medium 1A. The different blood c o m p o n e n t s - plasma, serum, RBC lysate and RBC g h o s t s - - w e r e then a d d e d in 0.3-ml aliquots to the purified l y m p h o c y t e cultures. Sodium selenite (Na2SeO3) (Alfa Products, Ventron Corporation, Beverly, Mass.) concentrations ranging from 1.58 X 10 -6 M to 7.90 X 10 -s M, 1 X 10 -4 M methyl methanesulfonate (MMS) (Eastman), 5 × 10 -4 M ethyl methanesulfonate (EMS) (Eastman), and 2 X 10 -s M N-hydroxy-2-acetylaminofluorene (N-OH-AAF) were also added to whole blood and purified l y m p h o c y t e cultures at this time when indicated. Incubation was continued for 19 + 1 h. Colchicine (4 #g/ml, final concentration) was added during the final 3 h of culture. Long-term cultures. An SV40 virus-transformed xeroderma pigmentosum cell line (XP12RO) was grown in MEM supplemented with 2 mM glutamine and 20% fetal calf serum (FCS). Penicillin (100 units/ml) and streptomycin (100
346 pg/ml) were also present in the medium. For an experiment newly subcultured cells were pre-incubated in a 7.5% CO2 atmosphere (37°C) for 2 4 - - 4 8 h depending on the initial cell density. At approximately 50% confluency the medium was removed and 10 ml of fresh medium was added along with 1 × 10 -s M BrdU. FdU and U were omitted as described by Wolff et al. [39]. Incubation was continued for 24 h (37°C) at which time the medium was again removed, and the cell sheet was washed with 10 ml of a sterile solution containing 8 g NaC1, 1 g glucose, 0.4 g KC1, and 0.35 g NaHCO3/100 ml. Fresh medium was added along with different Na2SeO3 concentrations and 0.75 ml RBC lysate when indicated. After a further 20 h at 37°C colchicine (4 pg/ml) was added, and the cells were harvested 4 h later. A long-term h u m a n lymphoblastoid cell line was grown in RPMI 1640 medium (GIBCO) supplemented with glutamine, FCS, and antibiotics as described for the XP12RO cell line. Following 24--48 h of pre-incubation in a 7.5% CO~ atmosphere (37°C} 1 × 10 -s M BrdU, 4 × 10 -~ M FdU and 6 × 10 -6 M U were added to the cultures. Incubation was continued for an additional 24 h. Excess BrdU, FdU and U were then removed by centrifuging (675 × g; 5 min) and washing the cells once with MEM. The washed cell pellet was resuspended in fresh medium, and incubation was continued for a further 19 h. Na2SeO3 and 0.75 ml RBC lysate were added during the final culture period as indicated. Colchicine (4 pg/ml) was added for the final 3 h of culture.
Harvesting procedure All culture types, including whole blood, purified lymphocytes, XP12RO, and lymphoblastoid cells, were harvested by the same technique except that XP12RO cells were first removed from the surface of the T-flasks by trypsinization (0.025% trypsin and 0.025% Na2EDTA). Harvesting was accomplished by centrifugation (675 × g ; 5 min), resuspension of the pellets in 0.075 M KC1 (25 min; 25°C), and fixation 3 times in 3 methanol: I glacial acetic acid. Airdried slide preparations were made on cold, detergent-cleaned microscope slides.
Staining procedure Chromosomes were differentially-stained using either the hot phosphate-Giemsa technique [12] or 33258 Hoechst [13]. For Giemsa differential staining slides were incubated in 1 M Na2HPO4 (pH 8.1; 15 min; 88°C), rinsed in 1 change of 1 M Na2HPO4 (pH 8.1; 25°C) and 1 change of deionized water, and stained with 2% Giemsa (pH 6.8) until adequate differential staining was achieved (usually 5--10 min). The slides were rinsed in water, air-dried, and m o u n t e d in Permount prior to light microscopic examination. 33258 Hoechst differential staining was accomplished using essentially the procedure of CraigHolmes and Shaw [5]. The slides were refixed in methanol: glacial acetic acid (3 : 1) for 30 min. After being totally air-dried the slides were placed in 95% ethanol for 30 min, 70% ethanol for 5 min, and deionized water for 5 min. The slides were briefly rinsed in half strength McIlvaine's citric acid--phosphate buffer made by mixing 0.1 M citric acid and 0.2 M Na2HPO4 to a pH of 4.5 and then diluting with an equal volume of deionized water. The slides were stained for 15 min with 33258 Hoechst at a concentration of 0.5 pg/ml in the above
347
buffer. After rinsing for a total of 10 min in 3 changes of deionized water, the slides were finally m o u n t e d in half strength McIlvaine's buffer (pH 5.5). Slides were stored overnight in the dark at room temperature before being scored for SCEs. SCE frequencies were determined using a Zeiss fluorescence microscope equipped with an HBO-200 watt mercury vapor lamp. BG12 excitor and combined 5 3 / 4 4 barrier filters were used. A total of 25 cells was scored for SCEs for each treatment regardless of the staining technique employed. Results Na2SeO3 was equally toxic to whole blood and purified lymphocyte cultures (Table 1). Both whole blood and lymphocyte cultures survived exposure to 1.58 X 10 -s M Na2SeO3, but no mitotic figures were present on slides of 7.90 X 10 -s M Na2SeO~-exposed cultures. As had been previously shown [23] exposure of whole blood cultures to high Na2SeO3 concentrations (7.90 X 10-6--1.58 X 10 -s M) resulted in a 3- to 4-fold increase in the observed average SCE frequencies. The differences between the control SCE frequencies (6--8 SCEs/cell) and those of Na2SeO3-treated cultures (19--25 SCEs/cell) were statistically significant (P < 0.001). Exposure of purified lymphocytes to these same Na2SeO3 concentrations resulted in SCE frequencies that did not differ from those of control cultures. Only in a single case (Table 1; Individual II, Experiment II, 1.58 X 10 -s M) was there an indication that Na2SeO3 might induce SCEs in purified lymphocytes, and the SCE frequency in this case was only slightly higher than the control SCE frequency and was much less than
TABLE
1
SODIUM SELENITE-INDUCED BLOOD AND FICOLL--HYPAQUE
Individual
Na2SeO 3 concentration (M)
Control 1.58 X 10 7.90 × 10 1.19 × 10 1.58 X 10 7.90 X 10 II
-6 -6 -5 -S -5
Control 1.58 X 10 -6 7.90 X 10 -6 1.19 X 10 -5 1.58 X 10 -5 7.90 X 10 -5
SISTER-CHROMATID EXCHANGE FREQUENCIES -- PURIFIED LYMPHOCYTE CULTURES
IN
WHOLE
S C E / c e l l (D +- S . E . ) Expt. I
Expt. II
Whole blood
Lymphocytes
Whole blood
Lymphocytes
8 . 2 4 +- 0 . 5 8 7.20 + 0.53 2 4 . 7 2 +_ 1 . 9 6 a ND NS Dead
8.24 7.24 8.44 ND 9.80 Dead
5.76 5.28 19.48 22.96 NS Dead
7.80 6.48 7.60 9.24 8.72 Dead
+ ± + + +
6.32 6.16 25.00 ND 18.72 I~ead
11.76 9.88 9.28 ND 10.24 Dead
9.60 9.36 9.68 9.48 11.84 ND
+- 0 . 6 7 ± 0.76 -+ 0 . 7 0 b -+ 1 . 0 7 b ± 0.87 b
± 0.66 ± 0.57 ± 1.95 a ± 2.07 a
+ 0.60 + 0.33 ± 0.58 b ± 0.56 b
± 0.66 b ± 0.71 b ± 0.73 b ± 0.95 b
+ 0.37 ± 0.46 +_ 1 . 9 3 a -+ 2 . 3 9 a
8 . 4 4 +- 0 . 5 3 9 . 8 0 +- 0 . 6 9 NS NS NS ND
ND, not done. N S , u n a b l e to s c o r e due to culture p r o b l e m or inability t o d i f f e r e n t i a l l y strain. a Significant d i f f e r e n c e b e t w e e n c o n t r o l and N a 2 S e O 3 - e x p o s e d c u l t u r e s ( P ~ 0 . 0 0 1 ) . b S i g n i f i c a n t d i f f e r e n c e b e t w e e n w h o l e b l o o d and purified l y m p h o c y t e s ( P 0.50). RBC lysate and RBC ghosts were prepared and added to purified l y m p h o c y t e cultures of two individuals (Table 4). Neither RBC lysate nor RBC ghosts altered the SCE frequency when they were added to l y m p h o c y t e cultures w i t h o u t Na2SeO3. A control level SCE frequency also resulted from exposure of l y m p h o c y t e cultures to RBC ghosts plus 7.90 X 10 -6 M Na2SeO3. When RBC lysate was added with Na2SeO3, however, the SCE frequency was significantly higher than the control level SCE frequency (P < 0.001) and was similar to the elevated SCE frequency observed following
TABLE 2 MMS-, EMS-, A N D N - O H - A A F - I N D U C E D SISTER-CHROMATID AND PURIFIED LYMPHOCYTE CULTURES Treatment
Control 1 X 10 -4 M MMS 5 X 10 4 M EMS 2 X 1 0 -5 M N - O H - A A F
EXCHANGES
S C E / c e l l (]J + S . E . ) Whole Blood
Purified lymphocytes
8.96 33.36 15.20 16.56
8.44 35.20 17.76 17.20
_+ 0 . 6 6 +- 1 . 4 5 + 0.96 -+ 0 . 8 9
+- 0 . 4 4 +- 1 . 1 1 +- 0 . 7 6 -+ 1 . 2 5 a
a A t o t a l o f 15 cells was scored; 25 cells were scored in all o t h e r cases.
IN W H O L E
BLOOD
349
TABLE
3
DETERMINATION OF BLOOD COMPONENT RESPONSIBLE FOR 7.90 × 10 -6 M Na2SeO3-EXPOSED WHOLE BLOOD CULTURES
THE
Sample cultured
Na2SeO 3 a
SCE/cell (p ± S.E.) Individual I
Whole blood Whole blood
-+
5.88 ± 0.40 16.73 ± 2.88 b
Lymphocytes
--
7.52 ± 0.75
Lymphocytes Lymphocytes
+ plasma + plasma
-+
6.64 ± 0.42 7.76 + 0.64
Lymphocytes Lymphocytes
+ serum + serum
-+
7.28 + 0.63 8.72 + 0.62
Lymphocytes Lymphocytes
+ RBC pellet + RBC pellet
-+
INDUCTION
OF
SCEs IN
8.20 ± 0.71 1 4 . 8 1 _+ 2 . 3 1 b
a 7 . 9 0 X 10 -6 M N a 2 S e O 3 w a s a d d e d d u r i n g f i n a l 1 9 h o f c u l t u r e ; +, p r e s e n t ; - - , a b s e n t . b S i g n i f i c a n t d i f f e r e n c e b e t w e e n S C E f r e q u e n c y o f c o n t r o l a n d t r e a t e d c u l t u r e ( P
Mutation Research, 54 ( 1 9 7 8 ) 3 4 3 - - 3 5 4 © E l s e v i e r / N o r t h - H o l l a n d B i o m e d i c a l Press
SISTER-CHROMATID EXCHANGE INDUCTION BY SODIUM SELENITE: DEPENDENCE ON THE PRESENCE OF RED BLOOD CELLS OR RED BLOOD CELL LYSATE *
J A M E S H. R A Y a n d L E W I S C. A L T E N B U R G
University o f Texas Health Science Center, Graduate School o f Biomedical Sciences, Medical Genetics Center, P.O. Box 20334, Astrodome Station, Houston, Texas 77025 (U.S.A.) ( R e c e i v e d 22 M a y 1 9 7 8 ) ( R e v i s i o n r e c e i v e d 11 A u g u s t 1 9 7 8 ) ( A c c e p t e d 22 A u g u s t 1 9 7 8 )
Summary Sodium selenite (Na2SeO3) sister-chromatid exchange (SCE) induction was studied in both short-term and long-term cell cultures. The ability of Na2SeO3 to induce SCEs was found to depend on the culture conditions employed. Concentrations of Na2SeO3 (7.90 × 10 -6 M and greater) that produced elevated SCE frequencies in whole blood cultures resulted in control level SCE frequencies (6--8 SCEs/cell) in Ficoll--Hypaque -- purified lymphocyte cultures. However, whole blood and purified lymphocyte cultures were equally sensitive to SCE induction by methyl methanesulfonate (MMS), ethyl methanesulfonate (EMS), and N-hydroxy-2-acetylaminofluorene (N-OH-AAF). Analysis of different whole blood components showed that the presence of red blood cells (RBCs), and specifically RBC lysate, was a prerequisite for Na2SeO3 SCE induction in purified lymphocyte cultures. The SCE frequencies of xeroderma pigmentosum (XP12RO) and normal human lymphoblastoid cell lines were also found to be unaffected by Na2SeO3 concentrations that produced elevated SCE frequencies in whole blood cultures. Incubation of these latter two cell types with Na2SeO3 and RBC lysate resulted in SCE frequencies comparable to those in Na2SeO3-exposed whole blood cultures.
* A preliminary report of this work was presented at the 9th annual m e e t i n g o f t h e Environmental M u t a g e n Society, March 9--13, 1978, San Francisco, CA (U.S.A.).
Abbreviations: BrdU, bromodeoxyuridine~ EMS, ethyl methanesulfonate; FCS, fetal calf serum~ FdU, fhiorodeoxyuridine; HBSS, Hanks balanced salt solution; His +. histidine; MEM, minimum essential medium~ MMS, methyl methanesulfonate: Na2EDTA , disodium etbylenediamine tetraacetate~ N-OH-AAF, Nohydroxy-2-acetylaminofluorene~ RBC(s), red b l o o d cell(s)~ SCE(s), sisterchromatid exchange(s); U, uridine;/~ +- S.E., mean -+ standard error.
344 Introduction Sister-chromatid exchange (SCE) induction [ 1,6,20--22,34,39 ], unscheduled thymidine incorporation [24,35--37], and His + revertant production [18] all serve as sensitive indicators of DNA damage resulting from the exposure of mammalian and bacterial cells to chemical carcinogens and mutagens. A compound that exhibits the first two of these characteristics [17,23] is sodium selenite (Na2SeO~). Selenium-induced tumors have also been reported, but the data were criticized for the lack of proper controls [25,32 for reviews]; consequently, Na2SeO3 is not presently considered to be a carcinogen. In fact it exhibits anticarcinogenic [4,7,9,26--29,31], antimutagenic [10], and anticlastogenic [30] properties when used in combination with carcinogens, mutagens, and clastogens. Yet when Na2SeO3 is used alone, it is clastogenic [19], induces SCEs [23], and causes a significant amount of unscheduled DNA synthesis following incubation with crude liver extract [17 ]. Incubation with crude liver extract results in the activation of a group of compounds known as procarcinogens. These compounds generally do not induce SCEs [20,34,38], do not cause unscheduled DNA synthesis [24,35-37], and are not mutagenic [18] in in vitro tests because the organisms and cell cultures are not capable of converting them to proximate and ultimate carcinogenic forms. Following in vitro incubation with crude liver extract [2 ], however, these compounds exhibit the characteristic properties of carcinogens and mutagens. Thus, the available evidence suggests that while Na2SeO3 is on the one hand an anticarcinogenic/antimutagenic/anticlastogenic agent, it may also be a procarcinogen which can be activated under suitable conditions. In view of the conflicting results that have been obtained as to the carcinogenic potential of Na2SeO3 further study of Na2SeO3 SCE induction appeared warranted. The results of the experiments reported here demonstrate that Na2SeO3 alone does not induce SCEs when added to most culture systems. However, co-incubation of Na2SeO3 with red blood cells or red blood cell lysate results in its "activation" so that elevated SCE frequencies are produced. Materials and methods
Culture conditions Whole blood fractionation. L y m p h o c y t e s were purified from whole blood by the Ficoll--Hypaque gradient separation technique of B o y u m [3] with certain modifications. Whole heparinized blood that had been diluted with an equal volume of sterile 0.9% NaC1 was layered onto Ficoll--Hypaque gradients consisting of 4 parts 8% Ficoll (Pharmacia) and 1 part 50% Hypaque (Winthrop Labs). The ratio of diluted blood to gradient was 4 : 1. After centrifugation (400 × g; 40 min; 25°C) the lymphocytes, which appeared as a cloudy ring at the interface between diluted blood and the Ficoll--Hypaque gradient, were removed with a sterile Pasteur pipette and placed in tubes partially filled with Hanks Balanced Salt Solution (HBSS) (GIBCO). The tubes were then totally filled with HBSS, and the l y m p h o c y t e s were pelleted b y centrifugation (900 r.p.m.; 15 min; 25°C). Following supernatant removal and l y m p h o c y t e resus-
345 pension in HBSS, the centrifugation step was repeated. The final l y m p h o c y t e pellet was resuspended in Chromosome Medium 1A (GIBCO). Several whole blood components were prepared for addition to the cell cultures. Plasma from heparinized blood was obtained b y removing the supernatant of centrifuged whole blood. For collection of serum from coagulated blood, unheparinized whole blood was left at room temperature for at least 3 h. The upper liquid layer was then centrifuged (675 Xg; 5 min) and the supernatant fraction removed. Red blood cell (RBC) samples were obtained from the RBC pellets of Ficoll--Hypaque purified lymphocytes. The pellets were washed twice with Minimum Essential Medium (MEM) (GIBCO) and resuspended in Chromosome Medium 1A to a final volume equal to the original blood volume. For RBC lysis a volume of sterile, deionized water (4°C) equal to the packed volume of RBCs from 30 ml of whole blood was added to a centrifuge tube, and the suspension was agitated with a glass rod for 2 min. Following centrifugation (10 000 Xg; 10 min; 4°C) the RBC lysate was dispensed in 1.0-ml aliquots and stored frozen until use. The pellet containing RBC ghosts was repeatedly washed with 30-ml quantities of MEM and resuspended in water until microscopic examination revealed an absence of intact cells. The final RBC ghost pellet was resuspended in 30 ml Chromosome Medium 1A and stored frozen in 1.0-ml aliquots. The RBC lysate and ghosts were thawed immediately before addition to the cultures. Blood cultures. Whole blood cultures were initiated b y adding 0.3 ml of whole heparinized blood to 4 ml Chromosome Medium 1A. L y m p h o c y t e cultures were prepared by adding approximately 1 X 106 purified lymphocytes to 4 ml Chromosome Medium 1A. Three healthy individuals served as blood donors. Individuals I and III were males; individual II was female. Purified l y m p h o c y t e and whole blood cultures were incubated at 37°C for 96 h. Whole RBCs (0.3 ml) were added, when desired, to the purified lymp h o c y t e cultures at the beginning of the incubation period. After 53 h of incubation 1 X 10 -s M bromodeoxyuridine (BrdU), 4 X 1 0 -7 M fluorodeoxyuridine (FdU), and 6 X 1 0 - 6 M uridine(U) were added to the cultures [14]. BrdU served as both a thymidine analogue and an inhibitor of ribonucleotide reductase while F d U and U were added to inhibit thymidylate synthetase activity and to offset possible adverse effects of FdU on R N A synthesis, respectively [11]. After another 24 h the cells were pelleted (675 × g; 5 min), washed with 4 ml MEM, and resuspended in 4 ml Chromosome Medium 1A. The different blood c o m p o n e n t s - plasma, serum, RBC lysate and RBC g h o s t s - - w e r e then a d d e d in 0.3-ml aliquots to the purified l y m p h o c y t e cultures. Sodium selenite (Na2SeO3) (Alfa Products, Ventron Corporation, Beverly, Mass.) concentrations ranging from 1.58 X 10 -6 M to 7.90 X 10 -s M, 1 X 10 -4 M methyl methanesulfonate (MMS) (Eastman), 5 × 10 -4 M ethyl methanesulfonate (EMS) (Eastman), and 2 X 10 -s M N-hydroxy-2-acetylaminofluorene (N-OH-AAF) were also added to whole blood and purified l y m p h o c y t e cultures at this time when indicated. Incubation was continued for 19 + 1 h. Colchicine (4 #g/ml, final concentration) was added during the final 3 h of culture. Long-term cultures. An SV40 virus-transformed xeroderma pigmentosum cell line (XP12RO) was grown in MEM supplemented with 2 mM glutamine and 20% fetal calf serum (FCS). Penicillin (100 units/ml) and streptomycin (100
346 pg/ml) were also present in the medium. For an experiment newly subcultured cells were pre-incubated in a 7.5% CO2 atmosphere (37°C) for 2 4 - - 4 8 h depending on the initial cell density. At approximately 50% confluency the medium was removed and 10 ml of fresh medium was added along with 1 × 10 -s M BrdU. FdU and U were omitted as described by Wolff et al. [39]. Incubation was continued for 24 h (37°C) at which time the medium was again removed, and the cell sheet was washed with 10 ml of a sterile solution containing 8 g NaC1, 1 g glucose, 0.4 g KC1, and 0.35 g NaHCO3/100 ml. Fresh medium was added along with different Na2SeO3 concentrations and 0.75 ml RBC lysate when indicated. After a further 20 h at 37°C colchicine (4 pg/ml) was added, and the cells were harvested 4 h later. A long-term h u m a n lymphoblastoid cell line was grown in RPMI 1640 medium (GIBCO) supplemented with glutamine, FCS, and antibiotics as described for the XP12RO cell line. Following 24--48 h of pre-incubation in a 7.5% CO~ atmosphere (37°C} 1 × 10 -s M BrdU, 4 × 10 -~ M FdU and 6 × 10 -6 M U were added to the cultures. Incubation was continued for an additional 24 h. Excess BrdU, FdU and U were then removed by centrifuging (675 × g; 5 min) and washing the cells once with MEM. The washed cell pellet was resuspended in fresh medium, and incubation was continued for a further 19 h. Na2SeO3 and 0.75 ml RBC lysate were added during the final culture period as indicated. Colchicine (4 pg/ml) was added for the final 3 h of culture.
Harvesting procedure All culture types, including whole blood, purified lymphocytes, XP12RO, and lymphoblastoid cells, were harvested by the same technique except that XP12RO cells were first removed from the surface of the T-flasks by trypsinization (0.025% trypsin and 0.025% Na2EDTA). Harvesting was accomplished by centrifugation (675 × g ; 5 min), resuspension of the pellets in 0.075 M KC1 (25 min; 25°C), and fixation 3 times in 3 methanol: I glacial acetic acid. Airdried slide preparations were made on cold, detergent-cleaned microscope slides.
Staining procedure Chromosomes were differentially-stained using either the hot phosphate-Giemsa technique [12] or 33258 Hoechst [13]. For Giemsa differential staining slides were incubated in 1 M Na2HPO4 (pH 8.1; 15 min; 88°C), rinsed in 1 change of 1 M Na2HPO4 (pH 8.1; 25°C) and 1 change of deionized water, and stained with 2% Giemsa (pH 6.8) until adequate differential staining was achieved (usually 5--10 min). The slides were rinsed in water, air-dried, and m o u n t e d in Permount prior to light microscopic examination. 33258 Hoechst differential staining was accomplished using essentially the procedure of CraigHolmes and Shaw [5]. The slides were refixed in methanol: glacial acetic acid (3 : 1) for 30 min. After being totally air-dried the slides were placed in 95% ethanol for 30 min, 70% ethanol for 5 min, and deionized water for 5 min. The slides were briefly rinsed in half strength McIlvaine's citric acid--phosphate buffer made by mixing 0.1 M citric acid and 0.2 M Na2HPO4 to a pH of 4.5 and then diluting with an equal volume of deionized water. The slides were stained for 15 min with 33258 Hoechst at a concentration of 0.5 pg/ml in the above
347
buffer. After rinsing for a total of 10 min in 3 changes of deionized water, the slides were finally m o u n t e d in half strength McIlvaine's buffer (pH 5.5). Slides were stored overnight in the dark at room temperature before being scored for SCEs. SCE frequencies were determined using a Zeiss fluorescence microscope equipped with an HBO-200 watt mercury vapor lamp. BG12 excitor and combined 5 3 / 4 4 barrier filters were used. A total of 25 cells was scored for SCEs for each treatment regardless of the staining technique employed. Results Na2SeO3 was equally toxic to whole blood and purified lymphocyte cultures (Table 1). Both whole blood and lymphocyte cultures survived exposure to 1.58 X 10 -s M Na2SeO3, but no mitotic figures were present on slides of 7.90 X 10 -s M Na2SeO~-exposed cultures. As had been previously shown [23] exposure of whole blood cultures to high Na2SeO3 concentrations (7.90 X 10-6--1.58 X 10 -s M) resulted in a 3- to 4-fold increase in the observed average SCE frequencies. The differences between the control SCE frequencies (6--8 SCEs/cell) and those of Na2SeO3-treated cultures (19--25 SCEs/cell) were statistically significant (P < 0.001). Exposure of purified lymphocytes to these same Na2SeO3 concentrations resulted in SCE frequencies that did not differ from those of control cultures. Only in a single case (Table 1; Individual II, Experiment II, 1.58 X 10 -s M) was there an indication that Na2SeO3 might induce SCEs in purified lymphocytes, and the SCE frequency in this case was only slightly higher than the control SCE frequency and was much less than
TABLE
1
SODIUM SELENITE-INDUCED BLOOD AND FICOLL--HYPAQUE
Individual
Na2SeO 3 concentration (M)
Control 1.58 X 10 7.90 × 10 1.19 × 10 1.58 X 10 7.90 X 10 II
-6 -6 -5 -S -5
Control 1.58 X 10 -6 7.90 X 10 -6 1.19 X 10 -5 1.58 X 10 -5 7.90 X 10 -5
SISTER-CHROMATID EXCHANGE FREQUENCIES -- PURIFIED LYMPHOCYTE CULTURES
IN
WHOLE
S C E / c e l l (D +- S . E . ) Expt. I
Expt. II
Whole blood
Lymphocytes
Whole blood
Lymphocytes
8 . 2 4 +- 0 . 5 8 7.20 + 0.53 2 4 . 7 2 +_ 1 . 9 6 a ND NS Dead
8.24 7.24 8.44 ND 9.80 Dead
5.76 5.28 19.48 22.96 NS Dead
7.80 6.48 7.60 9.24 8.72 Dead
+ ± + + +
6.32 6.16 25.00 ND 18.72 I~ead
11.76 9.88 9.28 ND 10.24 Dead
9.60 9.36 9.68 9.48 11.84 ND
+- 0 . 6 7 ± 0.76 -+ 0 . 7 0 b -+ 1 . 0 7 b ± 0.87 b
± 0.66 ± 0.57 ± 1.95 a ± 2.07 a
+ 0.60 + 0.33 ± 0.58 b ± 0.56 b
± 0.66 b ± 0.71 b ± 0.73 b ± 0.95 b
+ 0.37 ± 0.46 +_ 1 . 9 3 a -+ 2 . 3 9 a
8 . 4 4 +- 0 . 5 3 9 . 8 0 +- 0 . 6 9 NS NS NS ND
ND, not done. N S , u n a b l e to s c o r e due to culture p r o b l e m or inability t o d i f f e r e n t i a l l y strain. a Significant d i f f e r e n c e b e t w e e n c o n t r o l and N a 2 S e O 3 - e x p o s e d c u l t u r e s ( P ~ 0 . 0 0 1 ) . b S i g n i f i c a n t d i f f e r e n c e b e t w e e n w h o l e b l o o d and purified l y m p h o c y t e s ( P 0.50). RBC lysate and RBC ghosts were prepared and added to purified l y m p h o c y t e cultures of two individuals (Table 4). Neither RBC lysate nor RBC ghosts altered the SCE frequency when they were added to l y m p h o c y t e cultures w i t h o u t Na2SeO3. A control level SCE frequency also resulted from exposure of l y m p h o c y t e cultures to RBC ghosts plus 7.90 X 10 -6 M Na2SeO3. When RBC lysate was added with Na2SeO3, however, the SCE frequency was significantly higher than the control level SCE frequency (P < 0.001) and was similar to the elevated SCE frequency observed following
TABLE 2 MMS-, EMS-, A N D N - O H - A A F - I N D U C E D SISTER-CHROMATID AND PURIFIED LYMPHOCYTE CULTURES Treatment
Control 1 X 10 -4 M MMS 5 X 10 4 M EMS 2 X 1 0 -5 M N - O H - A A F
EXCHANGES
S C E / c e l l (]J + S . E . ) Whole Blood
Purified lymphocytes
8.96 33.36 15.20 16.56
8.44 35.20 17.76 17.20
_+ 0 . 6 6 +- 1 . 4 5 + 0.96 -+ 0 . 8 9
+- 0 . 4 4 +- 1 . 1 1 +- 0 . 7 6 -+ 1 . 2 5 a
a A t o t a l o f 15 cells was scored; 25 cells were scored in all o t h e r cases.
IN W H O L E
BLOOD
349
TABLE
3
DETERMINATION OF BLOOD COMPONENT RESPONSIBLE FOR 7.90 × 10 -6 M Na2SeO3-EXPOSED WHOLE BLOOD CULTURES
THE
Sample cultured
Na2SeO 3 a
SCE/cell (p ± S.E.) Individual I
Whole blood Whole blood
-+
5.88 ± 0.40 16.73 ± 2.88 b
Lymphocytes
--
7.52 ± 0.75
Lymphocytes Lymphocytes
+ plasma + plasma
-+
6.64 ± 0.42 7.76 + 0.64
Lymphocytes Lymphocytes
+ serum + serum
-+
7.28 + 0.63 8.72 + 0.62
Lymphocytes Lymphocytes
+ RBC pellet + RBC pellet
-+
INDUCTION
OF
SCEs IN
8.20 ± 0.71 1 4 . 8 1 _+ 2 . 3 1 b
a 7 . 9 0 X 10 -6 M N a 2 S e O 3 w a s a d d e d d u r i n g f i n a l 1 9 h o f c u l t u r e ; +, p r e s e n t ; - - , a b s e n t . b S i g n i f i c a n t d i f f e r e n c e b e t w e e n S C E f r e q u e n c y o f c o n t r o l a n d t r e a t e d c u l t u r e ( P
