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Characterization of DNA lesions produced by HgCl2 in cell culture systems
Chem.-Biol. Interactions, 49 (1984) 209--224
209
ElsevierScientificPublishersIreland Ltd.
CHARACTERIZATION OF CELL CULTURE SYSTEMS
DNA
ORAZIO COSTA*
T. CHRISTIE, STEVEN
CANTONI,
NELWYN
LESIONS
PRODUCED
BY HgCI2 IN
H. ROBISON
and M A X
Department of Pharmacology, University of Texas, Medical School at Houston, P.O. Box 20708, Houston, T X 77025 (U.S.A.)
(Received August 31st, 1983) (Revision received December 1st,1983) (Accepted December 2nd, 1983)
SUMMARY HgCI2 is extremely cytotoxic to Chinese hamster ovary {CHO) cells in culture since a 1-h exposure to a 75-~M concentration of this c o m p o u n d reduced cell plating efficiency to 0 and cell growth was completely inhibited at 7.5 ~M. The level of HgCI2 toxicity depended upon the culture incubation medium and has previously been shown to be inversely proportional to the extracellular concentration of metal chelating amino acids such as cysteine. Thus, HgCI2 toxicity in a minimal salts/glucose maintenance m e d i u m was about 10-fold greater than the toxicity in McCoy's culture medium. The HgCl2 toxicity in the latter m e d i u m was 3-fold greater than that in a - M E M which contains more of the metal chelating amino acids. W h e n cells were exposed to HgCl2 there was a rapid and pronounced induction of single strand breaks in the D N A at time intervals and concentrations that paralleled the cellular toxicity. The D N A damage was shown to be true single strand breaks and not alkaline sensitive sites or double strand breaks by a variety of techniques. Consistent with the toxicity of HgCI2, the D N A damage under an equivalent exposure situation was more pronounced in the salts/glucose than in the McCoy's m e d i u m and more striking in the latter medium than in a-MEM. Most of the single strand breaks occurred within 1 h of exposure to the metal. W e believe that the D N A damage caused by HgCI2 leads to cell death because the D N A single strand breaks are not readily repaired. D N A repair activity measured by CsCl density gradient techniques was elevated above the untreated levels at HgCl2 concentrations that produced little measurable binding of the metal to D N A or few single strand breaks assessed by the alkaline elution procedure. D N A repair activity decreased at HgCl2 *To whom correspondence should be sent. Abbreviations: CHO, Chinese hamster ovary; SDS, sodium dodecyl sulfate;SSF, strand scissionfactor. 0009-2797/84/$03.00 © 1984 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
210 concentrations that produced measurable DNA binding and single strand breaks. These irreversible interactions of HgC12 with DNA may be responsible for its cytotoxic action in cells.
Key words: Mercury toxicity -- DNA damage
INTRODUCTION
The cellular mechanism of Hg(II) toxicity is currently not well understood. Since this metal is extremely reactive as indicated by its low op value and high degree of chemical softness it readily forms coordinate-covalent bonds with a variety of biological molecules [1]. Thus, enzymes such as Na+/K ÷ ATPase that contain and require reduced sulfhydryl groups for enzymatic activity are severely inhibited by this metal ion [2]. Recent evidence indicates that Hg 2÷ is capable of causing the formation of oxygen radicals in cells [3], thus offering another potential source of cellular injury. In a sense free Hg 2÷ ions being so reactive could be considered as a radical species. These factors contribute to the high toxic activity displayed by Hg 2÷ at the cellular level. Previous studies have demonstrated that exposure of cultured CHO cells to HgC12-induced DNA single strand breaks [4] that correlated well with its ability to cause cell death [5]. The single strand breaks induced by other chemical and physical agents including metals which are not inhibitory to DNA repair were generally rapidly rejoined [6,7] but those caused by HgCl2 were not readily reversed since HgC12 has been shown to inhibit the repair of single strand breaks induced by X-rays [5]. Since irreversible DNA strand breaks are produced by HgC12 at very low concentrations that paralleled its cellular toxicity, it is likely that cell death may result from these lesions [ 5 ]. In the present study, we have focused upon the nature of the DNA lesion produced by HgC12 in cultured cells and have definitively characterized these lesions utilizing a number of techniques. We have also correlated the induction of these lesions with DNA repair activity and Hg 2+ binding to DNA. MATERIALS AND METHODS
Materials The radioisotopes [3H]thymidine (55 mCi/mmol) and ['4C]thymidine (58 mCi/mmol) were purchased from New England Nuclear Corporation (Boston, MA). The radionuclide [32p]orthophosphate was obtained from ICN radiochemicals (Irvine, CA). Ultrapure sucrose, sodium dodecyl sulfate (SDS), sodium dodecyl sarkosine, free acid EDTA, disodium EDTA and RNAse were all obtained from Sigma Chemical Company (St. Louis, MO). Tetrapropylammonium hydroxide (10% aqueous solution} was purchased
211 from RSA Chemical Company (Ardsley, NY). Polyvinylchloride filters were obtained from Millipore Corp. (Bedford, MA) and p o l y c a r b o n a t e filters were from Nucleopore (Pleasanton, CA). Proteinase K was purchased from MC/B chemicals (Cincinatti, OH). Bovine serum, McCoy's 5A medium and trypsin were purchased from Gibco, Inc. Liquiscint® was obtained from National Diagnostics (Somerville, NJ}.
Methods Growth inhibition studies. CHO cells used for c y t o t o x i c i t y studies were seeded at 1 X l 0 s cultured cells in 60-mm Petri dishes containing 5 ml of McCoy's 5A medium supplemented with 10% serum. Cells were incubated in media containing appropriate HgCl~ concentrations for selected time intervals. Following t r e a t m ent , the medium was removed. The cell monolayer was washed with 10 ml o f Puck's saline A (4 mM NaHCO3 (pH 7.2} containing 0.14 M NaC1, 5 mM KC1 and 5 mM dextrose) and scraped with a rubber policeman. Cell num be r was determined by using a Coulter particle counter. Colony forming assay. CHO cells in logarithmic growth were exposed to a selected range o f HgC12 concentrations for 1 h. The cells were rinsed twice with saline A, trypsinized, plated in 100-mm Petri dishes and allowed to form colonies. Colonies were fixed, stained with crystal violet and the n u m b e r of surviving colonies in each plate was expressed as a function of the cell n u m b e r plated to obtain measurements of plating efficiencies. Alkaline elution. The alkaline elution t echni que for analysis o f DNA strand breaks was performed, with some m i nor modifications, as described by Ko h n et al. [ 8, 9] . CHO cells were seeded at 1 X 106 cells per 100-mm Petri dish in 10 ml o f 10% serum-supplemented McCoy's 5A medium containing 0.02 uCi ml -~ of [~4C]thymidine and incubated for 24 h at 37°C. The medium was removed and 10 ml of medium containing 2.5 pg ml -~ of unlabeled t h y m i d i n e was added. The cells were incubated at 37°C for 6 h and subsequently t he cultures were treated with HgC12. Cells were rinsed once with saline A and 3 ml of 0.5% trypsin added for 3 min. Cells were then diluted in saline A and c o u n t e d in a Coulter particle counter. An aliquot containing 8.5 X l 0 s cells was diluted t o 20 ml with ice cold saline A and deposited on 25 mm p o l y c a r b o n a t e or polyvinylchloride filters (2.0 ~m pore size). The cells were then lysed with 5 ml of 2% SDS, 0.025 M EDTA (pH 10.1) ( p o l y c a r b o n a t e filters} or 0.2% sodium dodecyl sarkosine, 2 M NaC1, and 0.04 M EDTA (pH 10.1} (polyvinylchloride filters} and washed with 5 ml of 0.02 M EDTA. The lysates on the polyvinylchloride filters were additionally treated by adding 0.5 mg/ml proteinase K in lysis buffer for 60 min. Elution was achieved by adding 25 ml of a solution containing 0.02 M EDTA (free acid} plus 2% (final concentration} tetrapropylammo n iu m h y d r o x i d e (pH 12.2) ( pol ycarbonat e filters} or the same solution plus 0.1% SDS (polyvinylchloride filters} and eluting at a rate of 0.035 ml min -~. Fractions of approx. 2 ml were collected. The filters were then digested for 1 h at 60°C in 1 N HCI, and c o u n t e d in 10 ml of Liquiscint®.
212 Strand scission factor (SSF) was calculated from the alkaline elution patterns b y t h e following relationship: SSF = - l o g A/B, where A -- a m o u n t of DNA retained in the sixth fraction of unt reat ed sample and B = DNA retained in the sixth fraction o f the treated sample. The presence of DNA double strand breaks was investigated by neutral elution m e t h o d s p e r f o r m e d according to Bradley and K ohn [ 1 0 ] . A ppr oxi m at el y 3 × l 0 s [ ' 4 C ] t h y m i d i n e labeled cells were deposited on a polyvinylchloride filter and lysed as described in the alkaline elution m e t h o d . DNA was eluted with 0.2% SDS, 0.02 M EDTA {free acid) and t e t r a p r o p y l a m m o n i u m h y d r o x i d e at pH 9.6. Elution rate was 0.035 ml min -I with a fraction interval of 90 min and a total elution time o f 12 h. Nucleoid gradient sedimentation. DNA lesions were also analyzed using nucleoid gradient sedimentation as previously described by Cook and Brazell [ 1 1 ] . Cells were seeded into 60-mm Petri dishes (3 × l 0 s cells/dish) and labeled with 0.4 uCi/ml o f [ 3 H ] t h y m i d i n e for 18--22 h at 37°C. HgC12 was added to t he medium for 1 h at the desired c o n c e n t r a t i o n as indicated in Table I. The medium was removed following metal t r e a t m e n t and cells were washed once with 10 ml of ice cold saline A. The cells were t h e n scraped with a rubber policeman and resuspended in 5 ml of saline A. An aliquot o f cells was counted, and the remainder of t he cells were centrifuged at 3500 rev./min in a Beckman TJ-6 centrifuge. The cell pellet was resuspended in sufficient saline A so that a 50-pl aliquot contained 1.5 × l 0 s cells. Pref o r m e d 15--30% neutral sucrose gradients were overlayered with 1 5 0 p l o f a lysis buffer (2.6 M NaC1, 1.33 mM EDTA, 2.6 mM Tris, pH 8.0, and 10% Triton X-100). A 50-~1 aliquot of t he cell suspension was gently injected into t he lysis layer. The ratio of cell aliquot volume to lysis volume is critical, as the final NaC1 c o n c e n t r a t i o n must be maintained at 1.95 M to insure o p t i m u m deproteinization o f DNA. The cells were lysed in the dark for 15--20 min at r oom t e m p e r a t u r e and t he gradients were then centrifuged at 31 000 rev./min for 30 min at 22°C in a Beckman SW 50.1 rotor. The gradients were fractionated by piercing a hole in the b o t t o m of the t ube and collecting 10 dr op aliquots o n t o Whatman 3 MM paper strips. Determination o [ D N A repair. Equilibrium density gradient centrifugation was utilized to measure DNA repair synthesis [ 1 2 ] . CHO cells were grown in media containing 0.5 ~ Ci/ml [ 32p] o r t h o p h o s p h a t e for 48 h t o label parental DNA. The m edi um was removed, fresh medium w i t hout radiolabel was added and t h e cells incubated at 37°C for an additional 48 h. B r o m o d e o x y uridine (10 -s M) was added 1 h prior to metal t r e a t m e n t and cells were subsequently exposed to the metal for a total of 1 h. The extracellular metal was removed and cells were treated for 3 h with 5 pCi/ml o f [3H]deoxythymidine. The medium was then removed and t he cell sheet washed once with ice cold saline A. The cells were scraped with a rubber policeman and 2 ml o f a lysis buffer was added (10 mM Tris--HC1, pH 8.0, 1 mM EDTA, 0.5% SDS). The resulting viscous suspension was transferred to screw-top plastic tubes to which 75 pg/ml of proteinase K was added and the m i xt ure was incubated at 37°C for 24 h. The proteinase K solution was freshly prepared and self-digested for 1.5 h at 37°C prior t o use. After incubation, the
213
digest was passed through a 25 gauge needle twice and CsC1 added to a final density of 1.72 g/ml ( g I = 1.4015). CsC1 gradients were centrifuged at 32 000 rev./min for 72 h in a Beckman SW 50.1 rotor. Aliquots (20 t~l) of fractions were spotted onto Whatman 3 MM filter paper, washed in ice cold 10% trichloroacetic acid containing 100 mM sodium pyrophosphate, then in ethanol, dried and counted. Parental density peak fractions were pooled (0.7--1.0 ml) and 0.7 ml of 1 M K2HPO4 at pH 12.5 was added and this mixture was allowed to remain at room temperature for 10--15 min. This solution was then diluted to 5 ml with 10 mM Tris--HCl, 1 mM EDTA (pH 8.0) and CsC1 added to a density of 1.76 mg/ml. The resulting CsC1-DNA preparation was transferred to Oak Ridge type screw cap polycarbonate centrifuge tubes and centrifuged in a Beckman SW 50.1 rotor at 36 000 rev./min for 72 h at 20°C to permit gradient formation. Gradients were fractionated by pumping the solution out of the tube and onto filter paper strips. A small portion of every fifth fraction was retained for determination of the refractive index. Fractions were assayed for acid-insoluble radioactivity as described for the neutral CsCl gradients. Cross counting of 32p in the 3H channel was determined to be ~1%. DNA isolation and agarose gel electrophoresis. DNA was isolated from cell cultures immediately after metal treatment according to a modification of a previously published procedure [13]. The cell layer of each 10 cm culture dish was washed twice with isotonic saline before adding 4 ml of buffer A containing 50 pg/ml proteinase K and incubating at 37°C for 12--16 h. Buffer A contained 10 mM Tris--HC1 (pH 8.0}, 10 mM EDTA, 10 mM NaC1 and 0.5% SDS. The viscous lysate was transferred to conical centrifuge tubes. An equal volume of buffer B-saturated phenol was added and the tubes were gently rolled for 10 min. Buffer B was as buffer A except the Tris concentration was 500 mM. The aqueous phase was collected by centrifugation at 500 X g. After a second extraction, the nucleic acids were precipitated by addition of 2.3 vol. of ethanol. After chilling at - 2 0 ° C for 2 h or in a dry ice-ethanol bath for 10 min, the precipitate was collected by centrifugation at 20 000 × g for 10 min. The precipitate was solubilized in 4 mM Tris--HCl (pH 8.0), 1.0 mM NaCl and I mM EDTA, and then treated with RNAse (50 ug/ml) for 1.5 h. The digestion mixture was extracted with phenol as before and the DNA collected by precipitation with 70% ethanol. Binding o f Hg to the DNA. t-Ig binding to DNA was assessed as described in greater detail elsewhere [14]. Cells were exposed to 2°3HgC12 for 1 h at the concentrations indicated in Fig. 1. DNA was isolated by a gentle procedure (vide supra) and the radioactive Hg associated with the DNA was determined. RESULTS HgC12 produced a time- and concentration-dependent induction of DNA strand breaks in intact cells based upon alkaline elution analysis (Fig. 1).
214 Untreated
2s~4
~o~
A
~
ed
15'
30' _z
o,
\
!
u-
200~M/ 150pM 60' k~ 180' ELU TION TIME (hr)
Fig. 1. Concentration and early temperal effects of HgCl: on DNA strand breaks analyzed by alkaline elution. CHO cells were treated for 1 h with varying concentrations of HgCl: (A) or with a 100 ~M concentration of HgCl: for varying time intervals (B). Mixing experiments indicated that t he observed DNA lesions occurred in intact cells and were not pr oduced following lysis of cells since [3H]thymidine-labeled untreated cells mixed with [~4C]thymidine-labeled cells treated with 100 pM HgC12 for 1 h had an elution pattern equivalent t o u n tr eated cells. These observed DNA strand breaks could represent alkaline labile sites, double strand breaks or true single strand breaks since the elution techniques p e r f o r m e d under alkaline conditions did not distinguish among these possible lesions. Figure 2 demonstrates an analysis of whether HgCl~-induced alkaline labile sites relative to a positive cont rol (N-methyl N-nitrosourea). At either pH 12.1 or 12.6 the elution o f DNA from untreated or HgC12-treated cultures was n o t significantly altered by raising the pH of the elution buffer whereas when cells were treated with Nm e t h y l N-nitrosourea alkaline labile sites were detected by comparing the elution pattern at the two pH levels. An additional elution analysis o f the DNA from N - m e t hyl N-nitrosourea treated cells indicates an initial linearity that was followed by a sudden convex curvature after 4--6 h of elution indicating that single strand breaks were continually form ed by the chemical conversion of damaged sites sensitive to the alkaline environment (Fig. 2). In contrast, DNA elution profiles of HgCl:-treated cells were linear with respect to time (Fig. 2). T he refore HgC12-induced true DNA strand breaks and not alkaline labile sites under exposure conditions similar to
215 1.0
a
z.5 tuJ cr
Z a z3 0 I(o <
°I
E
LL
2
i f 3
6 ~ ELUTIC~ r=uE (hr)
12
ELUTION TIME (hr)
Fig. 2. Analysis of alkaline labile sites induced by HgCI~ or M N U (inset) in C H O cells. Untreated cells (o o, • ~,) or cells treated with either HgCl 2 or M N U (50 # M , 1 h, '. o, • • ) were analyzed for strand breaks at p H 12.1 (closed symbols) or p H 12.6 (open symbols).
those utilized in Fig. 1. The question of whether HgCl~-induced DNA double or single strand breaks in cultured cells was investigated by neutral elution studies and by agarose gel electrophoresIs. Figure 3 demonstrates that when DNA elution was conducted at pH 9.6 no strand breaks were evident with HgCl~ treatment whereas high doses of X-rays (5 K-fads and 10 K-rads) increased the DNA elution rate. These high doses of X-rays are known to cause DNA double strand breaks [ 1 0 , 1 5 ] . At pH 9.6 DNA remains double stranded and therefore only DNA double strand breal~s will increase the elution rates. The inset of Fig. 3 shows the characteristic enhancement of DNA elution at pH 12.1 obtained following a similar treatment with HgC12 since at this pH the DNA was denatured and the presence of single strand breaks affected the rate of DNA elution from the filters. Figure 4 shows an agarose gel electrophoresis analysis of DNA from untreated cells or DNA from cells treated with HgC12 or CaCrO+. At high concentrations following
216
1.0~ 8
~:~
~-
~
=
pH 9.6
\
"\
pH 12.1 .1 3
6
9
12
ELUTION TIME (hr)
Fig. 3. Analysis o f t h e i n d u c t i o n o f D N A d o u b l e s t r a n d breaks. DNA was eluted at pH 9.6 t o e x a m i n e the i n d u c t i o n o f d o u b l e s t r a n d breaks in u n t r e a t e d CHO cells (o o) or cells t r e a t e d with HgCl= (100 uM, 1 h, , :~: ). A positive c o n t r o l was included by e x p o s i n g cells t o X-rays (5 K rads, ~ ~ a n d 10 K rads ,~ .'.). The inset s h o w s the n o r m a l p a t t e r n o f D N A single s t r a n d breaks o b t a i n e d in u n t r e a t e d ceils (e • ) or cells t r e a t e d with HgCI 2 (= ~ ) w h e n D N A was e l u t e d at pH 12.1.
18 h of exposure, CaCrO4 (100 pM) resulted in double strand breaks due to its cellular toxicity; this was evident in the migration of DNA on the agarose gel shown in Fig. 4 where the DNA from two untreated populations of cells is compared with that from a population of cells treated with CaCrO4. In contrast, when cells were treated with HgC12 most of the DNA remained in a molecular weight form indicating t h a t if there were double strand breaks induced by HgCl~ they could n o t be detected by this procedure (Fig. 4 ). Most of the DNA single strand breaks occurring with HgCl: treatment of cells were evident within the first hour of exposure since the uptake of this metal into cells was very rapid [4] (Figs. 1 and 5). However, at time intervals greater than 1 h of HgC12 treatment, DNA-DNA cross-links were evident (Fig. 6). There was no detectable DNA-protein cross-linking produced by HgC12 since proteinase K treatment of cell lysate did not alter the DNA elution rate {Fig. 6). In Fig. 6, the DNA elution rate from cells treated
217 A
B
HgCl= [/~M] 25
50
100
CaCrO, [/JM] 0
;~
100
0
0
--24kb
--9.6
-6.6
Fig. 4. Analysis b y agarose gel e l e c t r o p h o r e s i s o f d o u b l e strand breaks o f DNA. Cells were e x p o s e d to the indicated c o n c e n t r a t i o n s o f HgCl 2 for 1 h (A) and t o CaCrO~ (B) for 18 h. DNA was isolated f r o m t h e cells and subjected t o agarose gel e l e c t r o p h o r e s i s as d e s c r i b e d in t h e M e t h o d s section. F r a g m e n t s o f ~. DNA were p r o d u c e d by Hind III digestion and included as size markers.
with 25 ~M HgCl2 for 5 h was only slightly greater than that observed after 1 h and therefore only the 1-h exposure is shown. The DNA single strand breaks induced by HgC12 were examined in the nucleoid system since this allowed an investigation of the direct effect of HgCl: on the DNA molecule itself (Table I). The principle of this m e t h o d is based on the fact t h a t introduction of strand breaks decreases the sedimentation rate of nucleoids (supercoiled DNA prepared by detergent lysis of cells}. The results were expressed as ratios between the distance travelled in sucrose gradients by nucleoids prepared from treated and untreated cells or by nucleoids treated in vitro with HgC12. Treatment of cells with 25--100 ~M HgCl~ for 1 h resulted in strand breakage in subsequentlyprepared nucleoids as indicated by the striking decrease in sedimentation velocity. Addition of HgC12 directly to isolated nucleoids also induced strand breaks in a concentration dependent fashion suggesting a direct action of the metal on the DNA. The ability of HgC12 to induce c y t o t o x i c i t y and DNA
ELUTION TIME(hr)
12h
3h 6h
Untreated
.2-
i
3{
8
9
10
'~ ELUTtON TIME (hr)
600 rads ',,
\
\
'\\ 600 rads: ", 25 pM.1 hr
HgCl 2
25yM.1 hr
Untreated
Fig. 6. Analysis o f D N A - D N A crosslinks i n d u c e d by HgCl~. F o l l o w i n g e x p o s u r e to 25 uM HgCI 2 for 1 or 5 h cells were lysed with (c . . . . ~)) or w i t h o u t (e • ) p r o t e i n a s e K digestion. D N A was a n a l y z e d by alkaline elution, as described in the M e t h o d s section in the absence or presence o f a test dose o f X-rays as indicated in the figure.
Fig. 5. Time d e p e n d e n t i n d u c t i o n o f DNA single s t r a n d breaks by HgCI 2. F o l l o w i n g e x p o s u r e o f cell cultures to 50 , M HgCl, for the time intervals s h o w n in t h e figure, D N A was a n a l y z e d by t h e alkaline elution p r o c e d u r e as described in t h e M e t h o d s section.
LI-
o< rr"
I.-
o
z.3
< Z D
D uJ Z.5 FLU rr.4
1C 9 ~-e~-'---e~--..e~
00
b~
219 TABLE I EFFECT OF HgCl 2 ON THE SEDIMENTATION RATE OF NUCLEOIDS FROM CHO CEI,LS Treatment
Concentration (aM)
Ratio a W h o l e cells b
Isolated nucleoids c
--
--
1
1
HgCl2 HgCl: HgC12 HgCI 2 HgCl:
25 50 100 250 500
0.935 0.734 0.540 0.300 --
-0.79 0.66 0.41 0.27
aRatio between the distance travelled by the nucleoids of HgCl2-treated cells and the distance travelled by the nucleoids of untreated cells. bWhole cells were treated with HgCl: for 1 h at the concentrations shown in the table. Nucleoids were isolated and their distance of migration in neutral sucrose gradients was determined. CNucleoids isolated from untreated cells were exposed to the indicated concentrations of HgCl: for 15 min in the dark at room temperature. The distance of migration of the nucleoids in neutral sucrose gradients was determined (vide supra). d a m a g e was highly d e p e n d e n t u p o n t h e e x t r a c e l l u l a r m e d i u m . Previous studies h a v e s h o w n t h a t t h e c y t o t o x i c i t y o f HgCl: a n d NiCl: d e p e n d to a great e x t e n t on t h e e x t r a c e l l u l a r levels o f m e t a l c h e l a t i n g a m i n o acids such as c y s t e i n e , since t h e s e agents i n h i b i t e d m e t a l u p t a k e [ 1 6 ] . In a g r e e m e n t w i t h t h e s e studies Fig. 7 s h o w s t h a t t h e level o f D N A single strand b r e a k s p r o d u c e d b y HgC12 also d e p e n d e d u p o n t h e cell c u l t u r e media. When C H O cells w e r e i n c u b a t e d in t h e s a l t / g l u c o s e m e d i u m t h a t c o n t a i n e d no m e t a l c h e l a t i n g a m i n o acids, t h e D N A d a m a g e was m o r e p r o n o u n c e d t h a n if cells w e r e i n c u b a t e d in M c C o y ' s m e d i u m or a-MEM. ~-MEM c o n t a i n e d m o r e m e t a l c h e l a t i n g a m i n o acids t h a n M c C o y ' s 5a m e d i u m . T a b l e II s h o w s t h e e f f e c t o f HgC12 o n t h e g r o w t h a n d p l a t i n g e f f i c i e n c y o f C H O cells in M c C o y ' s m e d i u m . Cell division was c o m p l e t e l y i n h i b i t e d at 7.5 t~M HgC12 while e x p o s u r e o f cells for 1 h t o 75 p M HgCl: resulted in c o m p l e t e cell killing. T h e c y t o t o x i c level o f HgCl: in salts/glucose m e d i u m can be a p p r o x i m a t e d b y dividing t h e c o n c e n t r a t i o n in M c C o y ' s m e d i u m p r o d u c i n g cell d e a t h b y 10 a n d t h e c y t o t o x i c i t y in a - M E M can be o b t a i n e d b y m u l t i p l y i n g t h e M c C o y ' s c y t o t o x i c level b y t h r e e [ 1 4 , 1 6 ] . Since HgCl: clearly p r o d u c e d lesions in t h e D N A we investigated t h e r e l a t i o n s h i p b e t w e e n t h e p r o d u c t i o n o f t h e s e lesions, its binding t o t h e D N A a n d t h e repair o f t h e s e lesions. D N A repair a c t i v i t y m e a s u r e d b y CsC1 d e n s i t y g r a d i e n t c e n t r i f u g a t i o n was o n l y evident at low e x p o s u r e c o n c e n t r a t i o n s o f HgC12 ( < 2 5 p M ) . A t t h e s e low c o n c e n t r a t i o n s , t h e r e was little d e t e c t a b l e D N A s t r a n d b r e a k a g e a n d b i n d i n g o f Hg. When t h e c o n c e n t r a t i o n o f HgCl: was increased a b o v e 25 tiM for I h, t h e r e was a striking loss o f repair a c t i v i t y ; h o w e v e r , s t r a n d b r e a k s a n d b i n d i n g of Hg t o D N A increased in a c o n c e n t r a t i o n d e p e n d e n t fashion (Fig. 8).
220
lil .9
Mc Co,~y
J
CI I..IJ Z.5 ,< I,U.,I n".4 < Z C~ Z.~ 0
Salts/Glucose Medium
l
lb 1'5 2'5
5b
160 260
560
HgCI2(PM)
Fig. 7. Analysis of DNA single strand breaks in CHO cells treated with HgCI2 for 1 h in three different culture media. Data were plotted as the fraction of D N A retained on the filters following nine hours of elution and as a function of the HgCI~ dose. Alkaline elution was performed as described in the Methods section. The salts/glucose maintenance medium consisted of 50 mM Hepes Buffer pH 7.2, 100 mM NaCI, 5 mM KCI, 5 mM glucose and 2 mM CaCI2. DISCUSSION T h e alkaline elution t e c h n i q u e represents a significant d e v e l o p m e n t in t h e analysis o f t h e effects o f chemical agents u p o n t h e D N A o f cells. This p r o c e d u r e is e x t r e m e l y sensitive and versatile in being able t o distinguish a m o n g multiple D N A lesions including strand breaks, D N A - D N A and DNAp r o t e i n crosslinks [ 9 ] . It c a n n o t , however, distinguish b e t w e e n D N A d o u b l e and single strand breaks and since D N A is eluted at an alkaline pH, strand breaks m a y be generated b y the alkaline e n v i r o n m e n t required t o d e n a t u r e and elute D N A f r o m t h e filters. T h e p r e s e n t s t u d y was u n d e r t a k e n t o definitively c h a r a c t e r i z e t h e D N A lesions p r o d u c e d b y HgCl:. Results show t h a t
221 T A B L E II EFFECT
OF
HgCl~ ON P L A T I N G E F F I C I E N C Y A N D GROWTH O F CHO C E L L S
HgCI 2 c o n c e n t r a t i o n (#M)
Plating e f f i c i e n c yb (% c o n t r o l s )
Cell g r o w t h c (% c o n t r o l s )
1 2.5 5 7.5 10 25 50 75
N.D. a N.D. N.D. 104 93.8 72.3 18.7 0
110 91 34 3
± 14 -* 7 *- 9 ± 1 0 N.D. N.D. N.D.
aNot d e t e r m i n e d . b C H O cells were i n c u b a t e d for 1 h with M c C o y ' s m e d i u m c o n t a i n i n g various c o n c e n t r a t i o n s o f HgCl 2. F o l l o w i n g this t r e a t m e n t cells were plated t o f o r m c o l o n i e s for 8 days as d e s c r i b e d in t h e M e t h o d s section. Cl0S CHO cells were i n c u b a t e d in M c C o y ' s m e d i u m c o n t a i n i n g d i f f e r e n t c o n c e n t r a t i o n s o f HgCl~ and, 24 h later, t h e cell n u m b e r o f each plate was d e t e r m i n e d w i t h a Coulter particle c o u n t e r .
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"gCI2(PM) Fig. 8. C o r r e l a t i o n o f t h e b i n d i n g o f HgCI 2 t o D N A w i t h its i n d u c t i o n o f DNA repair and single s t r a n d breaks. CHO cells were e x p o s e d t o HgCl 2 for 1 h at t h e c o n c e n t r a t i o n s s h o w n in t h e figure. D N A repair activity was assessed utilizing CsCl d e n s i t y gradient s e d i m e n t a t i o n as d e s c r i b e d in t h e M e t h o d s section. Binding o f Hg t o D N A and t h e induct i o n o f single s t r a n d breaks were assessed as d e s c r i b e d in t h e M e t h o d s section.
222 HgC12 induced true single strand breaks and not alkaline labile sites or double strand breaks in the DNA. The number of agents with known capability to induce double strand breaks in the DNA is quite small and generally these agents produce both single and double strand breaks [17]. However, a number of agents are capable of producing alkaline labile sites [18,19] and it is important to determine if the single strand breaks are due to the alkaline pH required to elute DNA. Such information may yield some understanding of the interaction of an agent with DNA and may allow correlations to be made between specific DNA lesions and oncogenic activity. Interestingly, HgCl2 did not induce alkaline labile sites while preliminary data demonstrates that CaCrO4 produces alkaline dependent strand breaks. CaCrO4 [20] as well as other metal carcinogenic agents such as certain nickel compounds [21] induced DNA-protein crosslinks, while HgCl~ did not produce this lesion. Organic carcinogenic agents such as benzo[a] pyrene [22] are known to also cause the formation of DNA-protein cross-links as well. HgC12, however, produces DNA-DNA crosslinks which with time progressively increased in extent. These crosslinks are probably due to the ability of HgCl2 to interact with the bases directly [5] while the single strand breaks may result from the production of oxygen radicals by HgC12 and also by its interaction with DNA bases [3]. Such radicals have been postulated to mediate the X-ray-induced DNA damage of cells. These studies illustrate basic differences in the way metal compounds interact with DNA in intact cells. A complete understanding of the specific mode of interaction of the metal with DNA will be enhanced by studying the specific lesions produced with adequate methodology to distinguish among the possible lesions. The DNA lesions produced by HgCl2 must be considered in a different way from the DNA lesions induced by other agents. For example the single strand breaks induced by nickel compounds and CaCrO4 are repaired [20,21] while the strand breaks induced with HgC12 are not readily repaired [5]. At a low concentration of HgCl2, where strand breaks could not be detected by alkaline elution and Hg binding to DNA was also below the level of detection, DNA repair activity was elevated. Perhaps the repair activity at these low concentrations of HgCl2 was activated due to the presence of undetectable DNA lesions, but the metal concentration was not sufficient to inhibit repair. At higher concentrations of HgC12 DNA repair activity was inhibited, resulting in the detection of both strand breaks and Hg-DNA adducts. Hg is an extremely reactive metal that has a high affinity for sulfhydryl groups such as those present in proteins but the binding sites contained in DNA bases would have a considerably lower affinity. However, due to the critical function of DNA in the cell, its concentration and the fact that repair enzymes are inhibited by HgCl~ and cannot mend the DNA lesions, cell death may result directly from these genetic effects. In fact Table II shows that a 1-h exposure to 50--75 gM HgCI2 results in a high percentage of cell death and at these concentrations strand breaks and Hg binding to DNA are considerable (Fig. 8). Injury to the cell membrane has been
223 p u r p o r t e d t o be t h e basis o f Hg 2÷ c y t o t o x i c action. Although it is difficult t o precisely e x t r a p o l a t e c o n c e n t r a t i o n s o f HgC12 t h a t p r o d u c e cell memb r a n e injury to t h e levels t h a t cause D N A lesions because o f differences in c u l t u r e media, lack o f Hg u p t a k e m e a s u r e m e n t s in o t h e r systems etc., t h e levels o f Hg 2÷ t h a t p r o d u c e DNA lesions are at least as low if n o t l o w e r t h a n t h o s e t h a t injure t h e cell m e m b r a n e [ 2 3 ] . Since Hg 2~ has X-ray like effects in being able t o p r o d u c e o x y g e n radicals in cells [ 3 ] , and d e p l e t e cellular r e d u c e d g l u t a t h i o n e levels [4] t h e D N A m u s t be c o n s i d e r e d a target site o f its t o x i c action. If i n d u c t i o n o f D N A lesions and active repair o f t h e s e lesions are i m p o r t a n t for m u t a g e n i c i t y or c a r c i n o g e n i c i t y o f a chemical agent, t h e n Hg m a y be e x p e c t e d t o have weak m u t a g e n i c activity at low c o n c e n t r a t i o n s b u t at higher c o n c e n t r a t i o n s w h e r e D N A repair activity was inhibited t h e r e should be less mutagenic activity. Additionally, the D N A lesions induced b y HgC12 m a y result in miscoding during DNA replicat i o n ; h o w e v e r HgC12 has been s h o w n t o inhibit cell growth specifically in S phase [24] and t h e r e f o r e miscoding during D N A replication must o c c u r at c o n c e n t r a t i o n s o f HgC12 t h a t allow this process t o p r o c e e d in o r d e r t o achieve a m u t a g e n i c response in a surviving cell. These mechanistic findings m a y help explain the low m u t a g e n i c / c a r c i n o g e n i c activity displayed b y HgC12 in a n u m b e r o f e x p e r i m e n t a l systems [ 2 5 ] . ACK NOWLEDG EMENTS This w o r k was s u p p o r t e d by G r a n t R - 8 0 8 0 4 8 f r o m t h e United States E n v i r o n m e n t a l P r o t e c t i o n Agency, by C o n t r a c t D E - A S 0 5 - 8 1 E R 6 0 0 1 6 f r o m t h e U n i t e d States D e p a r t m e n t o f Energy and b y National Institutes o f Health G r a n t CA 2 9 5 8 1 f r o m t h e National Cancer Institute. T h e Environm e n t a l P r o t e c t i o n A g e n c y does n o t necessarily e n d o r s e any c o m m e r i c a l p r o d u c t s used in this s t u d y and t h e c o n c l u s i o n s r e p r e s e n t t h e views o f t h e a u t h o r s and do n o t r e p r e s e n t t h e opinions, policies or r e c o m m e n d a t i o n o f the E n v i r o n m e n t a l P r o t e c t i o n Agency. T h e a u t h o r s t h a n k Ms. Linda H a y g o o d for secretarial assistance and J. Daniel Heck for critically reading this m a n u s c r i p t . REFERENCES 1 M.W. Williams, J.D. Hoeschele, J.E. Turner, K.B. Jaeobson, N.T. Christie, C.L. Paton, L.H. Smith, H.R. Witschi and E.H. Lee, Chemical softness and acute metal toxicity in mice and drosophila, Toxicol. Appl. Pharmacol., 63 (1982) 461. 2 B.L. Vallee and D.D. Ulmer, Biochemical Effects of mercury, cadmium and lead, Annu. Rev. Biochem., 41 (1972) 91. 3 0 . Cantoni, S.H. Robison, R.M. Evans, N.T. Christie, D.B. Drath and M. Costa, Possible involvement of superoxide free radicals in the HgCl: induced DNA damage in CHO cells, Fed. Proc., 42 (1983) 1135. 4 0 . Cantoni, R.M. Evans and M. Costa, Similarity in the acute cytotoxic response of mammalian cells to mercury II and X-rays: DNA damage and glutathione depletion, Biochim. Biophys. Res. Commun., 108 (1982) 614.
224 5 0 . Cantoni and M. Costa, Correlations of DNA strand breakage and their repair with cell survival following acute exposure to mercury (If) and X-rays, Mol. Pharmacol., 24 (1983) 84. 6 J.D. Chapman and C.J. Gillespie, Radiation-induced events and their time scale in mammalian cells, Adv. Radiat. Biol., 9 ( 1 9 8 l ) 143. 7 O. Cantoni and M. Costa, Characterization and mechanism of DNA damage induced by chromium (VI) and mercury (II) in cultured mammalian cells, Proc. Am. Assoc. Cancer Res., 24 (1983) 74. 8 K.W. Kohn, L.C. Erickson, R.A.G. Ewig and C.A. Friedman, Fractionation of DNA from mammalian cells by alkaline elution, Biochemistry, 15 (1976) 4629. 9 K.W. Kohn, R.A.G. Ewig, L.C. Erickson and L.A. Zwelling, Measurement of strand breaks and cross-links by alkaline elution, in: E. Friedberg and P. Hanawalt (Eds.), DNA Repair: A Laboratory Manual of Research Procedures, Marcel Dekker, New York, 1981, pp. 379--401. 10 M.O. Bradley and K.W. Kohn, X-ray induced DNA double strand breaks production and repair in mammalian cells as measured by neutral filter elution, Nucleic Acids Res., 7 (1979) 793. 11 P.R. Cook and J.A. Brazell, Detection and repair of single-strand breaks in nuclear DNA, Nature, 263 (1976) 679. 12 C.A. Smith, P.K. Cooper and P.C. Hanawalt, Measurement of repair replication by equilibrium sedimentation, in: E. Friedberg and P.C. Hanawalt (Eds.), DNA Repair: A Laboratory Manual of Research Procedures, Marcel Dekker, New York, 1981, pp. 289--305. 13 M. Gross-Bellard, P. Oudet and P. Chambon, Isolation of high-molecular weight DNA from mammalian cells, Eur. J. Biochem., 36 (1973) 32. 14 N.T. Christie, O. Cantoni, R.M. Evans, R.E. Meyn and M. Costa, Use of mammalian DNA repair deficient mutants to assess the effects of toxic metal compounds on DNA, Biochem. Pharmacol. { 1984) in press. 15 D.L. Dougle, C.J. Gillespie and J.D. Chapman, DNA strand breaks, repair, and survival in x-irradiated mammalian cells, Proc. Natl. Acad. Sci. U.S.A., 73 (1976)809. 16 M.P. Abbracchio, R.M. Evans, J.D. Heck, O. Cantoni and M. Costa, The regulation of ionic uptake and cytotoxicity by specific amino acids and serum components, Biol. Trace Elem. Res., 4 {1982) 289. 17 W.E. Ross and M.O. Bradley, DNA double strand breaks in mammalian cells after exposure to intercalating agents, Biochim. Biophys. Acta, 654 (1981) 129. 18 N.R. Drinkwater and E.C. Miller, Estimation of apurinic/apyrimidinic sites and phoshotriesters in deoxyribonucleic acid treated with electrophilic carcinogens and mutagens, Biochemistry, 19 (1980) 5087. 19 D.H. Phillips and P.C. Hanawalt, Alkalisensitive sites in DNA from human cells treated with ultraviolet light, l'-acetoxysagrole or l'-acetoxyestragole, Carcinogenesis, 3 (1982) 935. 20 M.J. Tsapakos, T.H. Hampton and K.W. Wetterhahn Jennette, The carcinogen chromate induces DNA cross-links in rat liver and kidney, J. Biol. Chem., 256 (1981) 3623. 21 R.B. Ciccarelli and K.E. Wetterhahn, Nickel distribution and DNA lesions induced in rat tissues by the carcinogen nickel carbonate, Cancer Res., 42 (1982) 3544. 22 C.M. Ireland, A. Eastman and E. Bresnick, DNA-protein crosslinks induced in a hamster tracheal epithelial cell line by benzo(a)pyrene, Biochem. Biophys. Res. Commun., 109 {1982) 1291. 23 E. Walum and H. Marchner, Effects of mercuric chloride on membrane integrity of cultured cells, Toxicol. Lett., 18 (1983) 89. 24 M. Costa, O. Cantoni, M. deMars and D.E. Swartzendruber, Toxic metals produce an S-phase-specific cell cycle block, Res. Commun. Chem. Pathol. Pharmacol., 38 (1983) 405. 25 A. Leonard, P. Jacquet and R.R. Lauwerys, Mutagenicity and teratogenicty of mercury compounds, Mutat. Res., 114 {1983) 1.
209
ElsevierScientificPublishersIreland Ltd.
CHARACTERIZATION OF CELL CULTURE SYSTEMS
DNA
ORAZIO COSTA*
T. CHRISTIE, STEVEN
CANTONI,
NELWYN
LESIONS
PRODUCED
BY HgCI2 IN
H. ROBISON
and M A X
Department of Pharmacology, University of Texas, Medical School at Houston, P.O. Box 20708, Houston, T X 77025 (U.S.A.)
(Received August 31st, 1983) (Revision received December 1st,1983) (Accepted December 2nd, 1983)
SUMMARY HgCI2 is extremely cytotoxic to Chinese hamster ovary {CHO) cells in culture since a 1-h exposure to a 75-~M concentration of this c o m p o u n d reduced cell plating efficiency to 0 and cell growth was completely inhibited at 7.5 ~M. The level of HgCI2 toxicity depended upon the culture incubation medium and has previously been shown to be inversely proportional to the extracellular concentration of metal chelating amino acids such as cysteine. Thus, HgCI2 toxicity in a minimal salts/glucose maintenance m e d i u m was about 10-fold greater than the toxicity in McCoy's culture medium. The HgCl2 toxicity in the latter m e d i u m was 3-fold greater than that in a - M E M which contains more of the metal chelating amino acids. W h e n cells were exposed to HgCl2 there was a rapid and pronounced induction of single strand breaks in the D N A at time intervals and concentrations that paralleled the cellular toxicity. The D N A damage was shown to be true single strand breaks and not alkaline sensitive sites or double strand breaks by a variety of techniques. Consistent with the toxicity of HgCI2, the D N A damage under an equivalent exposure situation was more pronounced in the salts/glucose than in the McCoy's m e d i u m and more striking in the latter medium than in a-MEM. Most of the single strand breaks occurred within 1 h of exposure to the metal. W e believe that the D N A damage caused by HgCI2 leads to cell death because the D N A single strand breaks are not readily repaired. D N A repair activity measured by CsCl density gradient techniques was elevated above the untreated levels at HgCl2 concentrations that produced little measurable binding of the metal to D N A or few single strand breaks assessed by the alkaline elution procedure. D N A repair activity decreased at HgCl2 *To whom correspondence should be sent. Abbreviations: CHO, Chinese hamster ovary; SDS, sodium dodecyl sulfate;SSF, strand scissionfactor. 0009-2797/84/$03.00 © 1984 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
210 concentrations that produced measurable DNA binding and single strand breaks. These irreversible interactions of HgC12 with DNA may be responsible for its cytotoxic action in cells.
Key words: Mercury toxicity -- DNA damage
INTRODUCTION
The cellular mechanism of Hg(II) toxicity is currently not well understood. Since this metal is extremely reactive as indicated by its low op value and high degree of chemical softness it readily forms coordinate-covalent bonds with a variety of biological molecules [1]. Thus, enzymes such as Na+/K ÷ ATPase that contain and require reduced sulfhydryl groups for enzymatic activity are severely inhibited by this metal ion [2]. Recent evidence indicates that Hg 2÷ is capable of causing the formation of oxygen radicals in cells [3], thus offering another potential source of cellular injury. In a sense free Hg 2÷ ions being so reactive could be considered as a radical species. These factors contribute to the high toxic activity displayed by Hg 2÷ at the cellular level. Previous studies have demonstrated that exposure of cultured CHO cells to HgC12-induced DNA single strand breaks [4] that correlated well with its ability to cause cell death [5]. The single strand breaks induced by other chemical and physical agents including metals which are not inhibitory to DNA repair were generally rapidly rejoined [6,7] but those caused by HgCl2 were not readily reversed since HgC12 has been shown to inhibit the repair of single strand breaks induced by X-rays [5]. Since irreversible DNA strand breaks are produced by HgC12 at very low concentrations that paralleled its cellular toxicity, it is likely that cell death may result from these lesions [ 5 ]. In the present study, we have focused upon the nature of the DNA lesion produced by HgC12 in cultured cells and have definitively characterized these lesions utilizing a number of techniques. We have also correlated the induction of these lesions with DNA repair activity and Hg 2+ binding to DNA. MATERIALS AND METHODS
Materials The radioisotopes [3H]thymidine (55 mCi/mmol) and ['4C]thymidine (58 mCi/mmol) were purchased from New England Nuclear Corporation (Boston, MA). The radionuclide [32p]orthophosphate was obtained from ICN radiochemicals (Irvine, CA). Ultrapure sucrose, sodium dodecyl sulfate (SDS), sodium dodecyl sarkosine, free acid EDTA, disodium EDTA and RNAse were all obtained from Sigma Chemical Company (St. Louis, MO). Tetrapropylammonium hydroxide (10% aqueous solution} was purchased
211 from RSA Chemical Company (Ardsley, NY). Polyvinylchloride filters were obtained from Millipore Corp. (Bedford, MA) and p o l y c a r b o n a t e filters were from Nucleopore (Pleasanton, CA). Proteinase K was purchased from MC/B chemicals (Cincinatti, OH). Bovine serum, McCoy's 5A medium and trypsin were purchased from Gibco, Inc. Liquiscint® was obtained from National Diagnostics (Somerville, NJ}.
Methods Growth inhibition studies. CHO cells used for c y t o t o x i c i t y studies were seeded at 1 X l 0 s cultured cells in 60-mm Petri dishes containing 5 ml of McCoy's 5A medium supplemented with 10% serum. Cells were incubated in media containing appropriate HgCl~ concentrations for selected time intervals. Following t r e a t m ent , the medium was removed. The cell monolayer was washed with 10 ml o f Puck's saline A (4 mM NaHCO3 (pH 7.2} containing 0.14 M NaC1, 5 mM KC1 and 5 mM dextrose) and scraped with a rubber policeman. Cell num be r was determined by using a Coulter particle counter. Colony forming assay. CHO cells in logarithmic growth were exposed to a selected range o f HgC12 concentrations for 1 h. The cells were rinsed twice with saline A, trypsinized, plated in 100-mm Petri dishes and allowed to form colonies. Colonies were fixed, stained with crystal violet and the n u m b e r of surviving colonies in each plate was expressed as a function of the cell n u m b e r plated to obtain measurements of plating efficiencies. Alkaline elution. The alkaline elution t echni que for analysis o f DNA strand breaks was performed, with some m i nor modifications, as described by Ko h n et al. [ 8, 9] . CHO cells were seeded at 1 X 106 cells per 100-mm Petri dish in 10 ml o f 10% serum-supplemented McCoy's 5A medium containing 0.02 uCi ml -~ of [~4C]thymidine and incubated for 24 h at 37°C. The medium was removed and 10 ml of medium containing 2.5 pg ml -~ of unlabeled t h y m i d i n e was added. The cells were incubated at 37°C for 6 h and subsequently t he cultures were treated with HgC12. Cells were rinsed once with saline A and 3 ml of 0.5% trypsin added for 3 min. Cells were then diluted in saline A and c o u n t e d in a Coulter particle counter. An aliquot containing 8.5 X l 0 s cells was diluted t o 20 ml with ice cold saline A and deposited on 25 mm p o l y c a r b o n a t e or polyvinylchloride filters (2.0 ~m pore size). The cells were then lysed with 5 ml of 2% SDS, 0.025 M EDTA (pH 10.1) ( p o l y c a r b o n a t e filters} or 0.2% sodium dodecyl sarkosine, 2 M NaC1, and 0.04 M EDTA (pH 10.1} (polyvinylchloride filters} and washed with 5 ml of 0.02 M EDTA. The lysates on the polyvinylchloride filters were additionally treated by adding 0.5 mg/ml proteinase K in lysis buffer for 60 min. Elution was achieved by adding 25 ml of a solution containing 0.02 M EDTA (free acid} plus 2% (final concentration} tetrapropylammo n iu m h y d r o x i d e (pH 12.2) ( pol ycarbonat e filters} or the same solution plus 0.1% SDS (polyvinylchloride filters} and eluting at a rate of 0.035 ml min -~. Fractions of approx. 2 ml were collected. The filters were then digested for 1 h at 60°C in 1 N HCI, and c o u n t e d in 10 ml of Liquiscint®.
212 Strand scission factor (SSF) was calculated from the alkaline elution patterns b y t h e following relationship: SSF = - l o g A/B, where A -- a m o u n t of DNA retained in the sixth fraction of unt reat ed sample and B = DNA retained in the sixth fraction o f the treated sample. The presence of DNA double strand breaks was investigated by neutral elution m e t h o d s p e r f o r m e d according to Bradley and K ohn [ 1 0 ] . A ppr oxi m at el y 3 × l 0 s [ ' 4 C ] t h y m i d i n e labeled cells were deposited on a polyvinylchloride filter and lysed as described in the alkaline elution m e t h o d . DNA was eluted with 0.2% SDS, 0.02 M EDTA {free acid) and t e t r a p r o p y l a m m o n i u m h y d r o x i d e at pH 9.6. Elution rate was 0.035 ml min -I with a fraction interval of 90 min and a total elution time o f 12 h. Nucleoid gradient sedimentation. DNA lesions were also analyzed using nucleoid gradient sedimentation as previously described by Cook and Brazell [ 1 1 ] . Cells were seeded into 60-mm Petri dishes (3 × l 0 s cells/dish) and labeled with 0.4 uCi/ml o f [ 3 H ] t h y m i d i n e for 18--22 h at 37°C. HgC12 was added to t he medium for 1 h at the desired c o n c e n t r a t i o n as indicated in Table I. The medium was removed following metal t r e a t m e n t and cells were washed once with 10 ml of ice cold saline A. The cells were t h e n scraped with a rubber policeman and resuspended in 5 ml of saline A. An aliquot o f cells was counted, and the remainder of t he cells were centrifuged at 3500 rev./min in a Beckman TJ-6 centrifuge. The cell pellet was resuspended in sufficient saline A so that a 50-pl aliquot contained 1.5 × l 0 s cells. Pref o r m e d 15--30% neutral sucrose gradients were overlayered with 1 5 0 p l o f a lysis buffer (2.6 M NaC1, 1.33 mM EDTA, 2.6 mM Tris, pH 8.0, and 10% Triton X-100). A 50-~1 aliquot of t he cell suspension was gently injected into t he lysis layer. The ratio of cell aliquot volume to lysis volume is critical, as the final NaC1 c o n c e n t r a t i o n must be maintained at 1.95 M to insure o p t i m u m deproteinization o f DNA. The cells were lysed in the dark for 15--20 min at r oom t e m p e r a t u r e and t he gradients were then centrifuged at 31 000 rev./min for 30 min at 22°C in a Beckman SW 50.1 rotor. The gradients were fractionated by piercing a hole in the b o t t o m of the t ube and collecting 10 dr op aliquots o n t o Whatman 3 MM paper strips. Determination o [ D N A repair. Equilibrium density gradient centrifugation was utilized to measure DNA repair synthesis [ 1 2 ] . CHO cells were grown in media containing 0.5 ~ Ci/ml [ 32p] o r t h o p h o s p h a t e for 48 h t o label parental DNA. The m edi um was removed, fresh medium w i t hout radiolabel was added and t h e cells incubated at 37°C for an additional 48 h. B r o m o d e o x y uridine (10 -s M) was added 1 h prior to metal t r e a t m e n t and cells were subsequently exposed to the metal for a total of 1 h. The extracellular metal was removed and cells were treated for 3 h with 5 pCi/ml o f [3H]deoxythymidine. The medium was then removed and t he cell sheet washed once with ice cold saline A. The cells were scraped with a rubber policeman and 2 ml o f a lysis buffer was added (10 mM Tris--HC1, pH 8.0, 1 mM EDTA, 0.5% SDS). The resulting viscous suspension was transferred to screw-top plastic tubes to which 75 pg/ml of proteinase K was added and the m i xt ure was incubated at 37°C for 24 h. The proteinase K solution was freshly prepared and self-digested for 1.5 h at 37°C prior t o use. After incubation, the
213
digest was passed through a 25 gauge needle twice and CsC1 added to a final density of 1.72 g/ml ( g I = 1.4015). CsC1 gradients were centrifuged at 32 000 rev./min for 72 h in a Beckman SW 50.1 rotor. Aliquots (20 t~l) of fractions were spotted onto Whatman 3 MM filter paper, washed in ice cold 10% trichloroacetic acid containing 100 mM sodium pyrophosphate, then in ethanol, dried and counted. Parental density peak fractions were pooled (0.7--1.0 ml) and 0.7 ml of 1 M K2HPO4 at pH 12.5 was added and this mixture was allowed to remain at room temperature for 10--15 min. This solution was then diluted to 5 ml with 10 mM Tris--HCl, 1 mM EDTA (pH 8.0) and CsC1 added to a density of 1.76 mg/ml. The resulting CsC1-DNA preparation was transferred to Oak Ridge type screw cap polycarbonate centrifuge tubes and centrifuged in a Beckman SW 50.1 rotor at 36 000 rev./min for 72 h at 20°C to permit gradient formation. Gradients were fractionated by pumping the solution out of the tube and onto filter paper strips. A small portion of every fifth fraction was retained for determination of the refractive index. Fractions were assayed for acid-insoluble radioactivity as described for the neutral CsCl gradients. Cross counting of 32p in the 3H channel was determined to be ~1%. DNA isolation and agarose gel electrophoresis. DNA was isolated from cell cultures immediately after metal treatment according to a modification of a previously published procedure [13]. The cell layer of each 10 cm culture dish was washed twice with isotonic saline before adding 4 ml of buffer A containing 50 pg/ml proteinase K and incubating at 37°C for 12--16 h. Buffer A contained 10 mM Tris--HC1 (pH 8.0}, 10 mM EDTA, 10 mM NaC1 and 0.5% SDS. The viscous lysate was transferred to conical centrifuge tubes. An equal volume of buffer B-saturated phenol was added and the tubes were gently rolled for 10 min. Buffer B was as buffer A except the Tris concentration was 500 mM. The aqueous phase was collected by centrifugation at 500 X g. After a second extraction, the nucleic acids were precipitated by addition of 2.3 vol. of ethanol. After chilling at - 2 0 ° C for 2 h or in a dry ice-ethanol bath for 10 min, the precipitate was collected by centrifugation at 20 000 × g for 10 min. The precipitate was solubilized in 4 mM Tris--HCl (pH 8.0), 1.0 mM NaCl and I mM EDTA, and then treated with RNAse (50 ug/ml) for 1.5 h. The digestion mixture was extracted with phenol as before and the DNA collected by precipitation with 70% ethanol. Binding o f Hg to the DNA. t-Ig binding to DNA was assessed as described in greater detail elsewhere [14]. Cells were exposed to 2°3HgC12 for 1 h at the concentrations indicated in Fig. 1. DNA was isolated by a gentle procedure (vide supra) and the radioactive Hg associated with the DNA was determined. RESULTS HgC12 produced a time- and concentration-dependent induction of DNA strand breaks in intact cells based upon alkaline elution analysis (Fig. 1).
214 Untreated
2s~4
~o~
A
~
ed
15'
30' _z
o,
\
!
u-
200~M/ 150pM 60' k~ 180' ELU TION TIME (hr)
Fig. 1. Concentration and early temperal effects of HgCl: on DNA strand breaks analyzed by alkaline elution. CHO cells were treated for 1 h with varying concentrations of HgCl: (A) or with a 100 ~M concentration of HgCl: for varying time intervals (B). Mixing experiments indicated that t he observed DNA lesions occurred in intact cells and were not pr oduced following lysis of cells since [3H]thymidine-labeled untreated cells mixed with [~4C]thymidine-labeled cells treated with 100 pM HgC12 for 1 h had an elution pattern equivalent t o u n tr eated cells. These observed DNA strand breaks could represent alkaline labile sites, double strand breaks or true single strand breaks since the elution techniques p e r f o r m e d under alkaline conditions did not distinguish among these possible lesions. Figure 2 demonstrates an analysis of whether HgCl~-induced alkaline labile sites relative to a positive cont rol (N-methyl N-nitrosourea). At either pH 12.1 or 12.6 the elution o f DNA from untreated or HgC12-treated cultures was n o t significantly altered by raising the pH of the elution buffer whereas when cells were treated with Nm e t h y l N-nitrosourea alkaline labile sites were detected by comparing the elution pattern at the two pH levels. An additional elution analysis o f the DNA from N - m e t hyl N-nitrosourea treated cells indicates an initial linearity that was followed by a sudden convex curvature after 4--6 h of elution indicating that single strand breaks were continually form ed by the chemical conversion of damaged sites sensitive to the alkaline environment (Fig. 2). In contrast, DNA elution profiles of HgCl:-treated cells were linear with respect to time (Fig. 2). T he refore HgC12-induced true DNA strand breaks and not alkaline labile sites under exposure conditions similar to
215 1.0
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E
LL
2
i f 3
6 ~ ELUTIC~ r=uE (hr)
12
ELUTION TIME (hr)
Fig. 2. Analysis of alkaline labile sites induced by HgCI~ or M N U (inset) in C H O cells. Untreated cells (o o, • ~,) or cells treated with either HgCl 2 or M N U (50 # M , 1 h, '. o, • • ) were analyzed for strand breaks at p H 12.1 (closed symbols) or p H 12.6 (open symbols).
those utilized in Fig. 1. The question of whether HgCl~-induced DNA double or single strand breaks in cultured cells was investigated by neutral elution studies and by agarose gel electrophoresIs. Figure 3 demonstrates that when DNA elution was conducted at pH 9.6 no strand breaks were evident with HgCl~ treatment whereas high doses of X-rays (5 K-fads and 10 K-rads) increased the DNA elution rate. These high doses of X-rays are known to cause DNA double strand breaks [ 1 0 , 1 5 ] . At pH 9.6 DNA remains double stranded and therefore only DNA double strand breal~s will increase the elution rates. The inset of Fig. 3 shows the characteristic enhancement of DNA elution at pH 12.1 obtained following a similar treatment with HgC12 since at this pH the DNA was denatured and the presence of single strand breaks affected the rate of DNA elution from the filters. Figure 4 shows an agarose gel electrophoresis analysis of DNA from untreated cells or DNA from cells treated with HgC12 or CaCrO+. At high concentrations following
216
1.0~ 8
~:~
~-
~
=
pH 9.6
\
"\
pH 12.1 .1 3
6
9
12
ELUTION TIME (hr)
Fig. 3. Analysis o f t h e i n d u c t i o n o f D N A d o u b l e s t r a n d breaks. DNA was eluted at pH 9.6 t o e x a m i n e the i n d u c t i o n o f d o u b l e s t r a n d breaks in u n t r e a t e d CHO cells (o o) or cells t r e a t e d with HgCl= (100 uM, 1 h, , :~: ). A positive c o n t r o l was included by e x p o s i n g cells t o X-rays (5 K rads, ~ ~ a n d 10 K rads ,~ .'.). The inset s h o w s the n o r m a l p a t t e r n o f D N A single s t r a n d breaks o b t a i n e d in u n t r e a t e d ceils (e • ) or cells t r e a t e d with HgCI 2 (= ~ ) w h e n D N A was e l u t e d at pH 12.1.
18 h of exposure, CaCrO4 (100 pM) resulted in double strand breaks due to its cellular toxicity; this was evident in the migration of DNA on the agarose gel shown in Fig. 4 where the DNA from two untreated populations of cells is compared with that from a population of cells treated with CaCrO4. In contrast, when cells were treated with HgC12 most of the DNA remained in a molecular weight form indicating t h a t if there were double strand breaks induced by HgCl~ they could n o t be detected by this procedure (Fig. 4 ). Most of the DNA single strand breaks occurring with HgCl: treatment of cells were evident within the first hour of exposure since the uptake of this metal into cells was very rapid [4] (Figs. 1 and 5). However, at time intervals greater than 1 h of HgC12 treatment, DNA-DNA cross-links were evident (Fig. 6). There was no detectable DNA-protein cross-linking produced by HgC12 since proteinase K treatment of cell lysate did not alter the DNA elution rate {Fig. 6). In Fig. 6, the DNA elution rate from cells treated
217 A
B
HgCl= [/~M] 25
50
100
CaCrO, [/JM] 0
;~
100
0
0
--24kb
--9.6
-6.6
Fig. 4. Analysis b y agarose gel e l e c t r o p h o r e s i s o f d o u b l e strand breaks o f DNA. Cells were e x p o s e d to the indicated c o n c e n t r a t i o n s o f HgCl 2 for 1 h (A) and t o CaCrO~ (B) for 18 h. DNA was isolated f r o m t h e cells and subjected t o agarose gel e l e c t r o p h o r e s i s as d e s c r i b e d in t h e M e t h o d s section. F r a g m e n t s o f ~. DNA were p r o d u c e d by Hind III digestion and included as size markers.
with 25 ~M HgCl2 for 5 h was only slightly greater than that observed after 1 h and therefore only the 1-h exposure is shown. The DNA single strand breaks induced by HgC12 were examined in the nucleoid system since this allowed an investigation of the direct effect of HgCl: on the DNA molecule itself (Table I). The principle of this m e t h o d is based on the fact t h a t introduction of strand breaks decreases the sedimentation rate of nucleoids (supercoiled DNA prepared by detergent lysis of cells}. The results were expressed as ratios between the distance travelled in sucrose gradients by nucleoids prepared from treated and untreated cells or by nucleoids treated in vitro with HgC12. Treatment of cells with 25--100 ~M HgCl~ for 1 h resulted in strand breakage in subsequentlyprepared nucleoids as indicated by the striking decrease in sedimentation velocity. Addition of HgC12 directly to isolated nucleoids also induced strand breaks in a concentration dependent fashion suggesting a direct action of the metal on the DNA. The ability of HgC12 to induce c y t o t o x i c i t y and DNA
ELUTION TIME(hr)
12h
3h 6h
Untreated
.2-
i
3{
8
9
10
'~ ELUTtON TIME (hr)
600 rads ',,
\
\
'\\ 600 rads: ", 25 pM.1 hr
HgCl 2
25yM.1 hr
Untreated
Fig. 6. Analysis o f D N A - D N A crosslinks i n d u c e d by HgCl~. F o l l o w i n g e x p o s u r e to 25 uM HgCI 2 for 1 or 5 h cells were lysed with (c . . . . ~)) or w i t h o u t (e • ) p r o t e i n a s e K digestion. D N A was a n a l y z e d by alkaline elution, as described in the M e t h o d s section in the absence or presence o f a test dose o f X-rays as indicated in the figure.
Fig. 5. Time d e p e n d e n t i n d u c t i o n o f DNA single s t r a n d breaks by HgCI 2. F o l l o w i n g e x p o s u r e o f cell cultures to 50 , M HgCl, for the time intervals s h o w n in t h e figure, D N A was a n a l y z e d by t h e alkaline elution p r o c e d u r e as described in t h e M e t h o d s section.
LI-
o< rr"
I.-
o
z.3
< Z D
D uJ Z.5 FLU rr.4
1C 9 ~-e~-'---e~--..e~
00
b~
219 TABLE I EFFECT OF HgCl 2 ON THE SEDIMENTATION RATE OF NUCLEOIDS FROM CHO CEI,LS Treatment
Concentration (aM)
Ratio a W h o l e cells b
Isolated nucleoids c
--
--
1
1
HgCl2 HgCl: HgC12 HgCI 2 HgCl:
25 50 100 250 500
0.935 0.734 0.540 0.300 --
-0.79 0.66 0.41 0.27
aRatio between the distance travelled by the nucleoids of HgCl2-treated cells and the distance travelled by the nucleoids of untreated cells. bWhole cells were treated with HgCl: for 1 h at the concentrations shown in the table. Nucleoids were isolated and their distance of migration in neutral sucrose gradients was determined. CNucleoids isolated from untreated cells were exposed to the indicated concentrations of HgCl: for 15 min in the dark at room temperature. The distance of migration of the nucleoids in neutral sucrose gradients was determined (vide supra). d a m a g e was highly d e p e n d e n t u p o n t h e e x t r a c e l l u l a r m e d i u m . Previous studies h a v e s h o w n t h a t t h e c y t o t o x i c i t y o f HgCl: a n d NiCl: d e p e n d to a great e x t e n t on t h e e x t r a c e l l u l a r levels o f m e t a l c h e l a t i n g a m i n o acids such as c y s t e i n e , since t h e s e agents i n h i b i t e d m e t a l u p t a k e [ 1 6 ] . In a g r e e m e n t w i t h t h e s e studies Fig. 7 s h o w s t h a t t h e level o f D N A single strand b r e a k s p r o d u c e d b y HgC12 also d e p e n d e d u p o n t h e cell c u l t u r e media. When C H O cells w e r e i n c u b a t e d in t h e s a l t / g l u c o s e m e d i u m t h a t c o n t a i n e d no m e t a l c h e l a t i n g a m i n o acids, t h e D N A d a m a g e was m o r e p r o n o u n c e d t h a n if cells w e r e i n c u b a t e d in M c C o y ' s m e d i u m or a-MEM. ~-MEM c o n t a i n e d m o r e m e t a l c h e l a t i n g a m i n o acids t h a n M c C o y ' s 5a m e d i u m . T a b l e II s h o w s t h e e f f e c t o f HgC12 o n t h e g r o w t h a n d p l a t i n g e f f i c i e n c y o f C H O cells in M c C o y ' s m e d i u m . Cell division was c o m p l e t e l y i n h i b i t e d at 7.5 t~M HgC12 while e x p o s u r e o f cells for 1 h t o 75 p M HgCl: resulted in c o m p l e t e cell killing. T h e c y t o t o x i c level o f HgCl: in salts/glucose m e d i u m can be a p p r o x i m a t e d b y dividing t h e c o n c e n t r a t i o n in M c C o y ' s m e d i u m p r o d u c i n g cell d e a t h b y 10 a n d t h e c y t o t o x i c i t y in a - M E M can be o b t a i n e d b y m u l t i p l y i n g t h e M c C o y ' s c y t o t o x i c level b y t h r e e [ 1 4 , 1 6 ] . Since HgCl: clearly p r o d u c e d lesions in t h e D N A we investigated t h e r e l a t i o n s h i p b e t w e e n t h e p r o d u c t i o n o f t h e s e lesions, its binding t o t h e D N A a n d t h e repair o f t h e s e lesions. D N A repair a c t i v i t y m e a s u r e d b y CsC1 d e n s i t y g r a d i e n t c e n t r i f u g a t i o n was o n l y evident at low e x p o s u r e c o n c e n t r a t i o n s o f HgC12 ( < 2 5 p M ) . A t t h e s e low c o n c e n t r a t i o n s , t h e r e was little d e t e c t a b l e D N A s t r a n d b r e a k a g e a n d b i n d i n g o f Hg. When t h e c o n c e n t r a t i o n o f HgCl: was increased a b o v e 25 tiM for I h, t h e r e was a striking loss o f repair a c t i v i t y ; h o w e v e r , s t r a n d b r e a k s a n d b i n d i n g of Hg t o D N A increased in a c o n c e n t r a t i o n d e p e n d e n t fashion (Fig. 8).
220
lil .9
Mc Co,~y
J
CI I..IJ Z.5 ,< I,U.,I n".4 < Z C~ Z.~ 0
Salts/Glucose Medium
l
lb 1'5 2'5
5b
160 260
560
HgCI2(PM)
Fig. 7. Analysis of DNA single strand breaks in CHO cells treated with HgCI2 for 1 h in three different culture media. Data were plotted as the fraction of D N A retained on the filters following nine hours of elution and as a function of the HgCI~ dose. Alkaline elution was performed as described in the Methods section. The salts/glucose maintenance medium consisted of 50 mM Hepes Buffer pH 7.2, 100 mM NaCI, 5 mM KCI, 5 mM glucose and 2 mM CaCI2. DISCUSSION T h e alkaline elution t e c h n i q u e represents a significant d e v e l o p m e n t in t h e analysis o f t h e effects o f chemical agents u p o n t h e D N A o f cells. This p r o c e d u r e is e x t r e m e l y sensitive and versatile in being able t o distinguish a m o n g multiple D N A lesions including strand breaks, D N A - D N A and DNAp r o t e i n crosslinks [ 9 ] . It c a n n o t , however, distinguish b e t w e e n D N A d o u b l e and single strand breaks and since D N A is eluted at an alkaline pH, strand breaks m a y be generated b y the alkaline e n v i r o n m e n t required t o d e n a t u r e and elute D N A f r o m t h e filters. T h e p r e s e n t s t u d y was u n d e r t a k e n t o definitively c h a r a c t e r i z e t h e D N A lesions p r o d u c e d b y HgCl:. Results show t h a t
221 T A B L E II EFFECT
OF
HgCl~ ON P L A T I N G E F F I C I E N C Y A N D GROWTH O F CHO C E L L S
HgCI 2 c o n c e n t r a t i o n (#M)
Plating e f f i c i e n c yb (% c o n t r o l s )
Cell g r o w t h c (% c o n t r o l s )
1 2.5 5 7.5 10 25 50 75
N.D. a N.D. N.D. 104 93.8 72.3 18.7 0
110 91 34 3
± 14 -* 7 *- 9 ± 1 0 N.D. N.D. N.D.
aNot d e t e r m i n e d . b C H O cells were i n c u b a t e d for 1 h with M c C o y ' s m e d i u m c o n t a i n i n g various c o n c e n t r a t i o n s o f HgCl 2. F o l l o w i n g this t r e a t m e n t cells were plated t o f o r m c o l o n i e s for 8 days as d e s c r i b e d in t h e M e t h o d s section. Cl0S CHO cells were i n c u b a t e d in M c C o y ' s m e d i u m c o n t a i n i n g d i f f e r e n t c o n c e n t r a t i o n s o f HgCl~ and, 24 h later, t h e cell n u m b e r o f each plate was d e t e r m i n e d w i t h a Coulter particle c o u n t e r .
.
,
!
,
|
i
.6
2
.5
l(J
8
nr
m
>*
:.z
0.25 =1
.1 i
0
20
I
40
I
60
i
80
I
|
100
"gCI2(PM) Fig. 8. C o r r e l a t i o n o f t h e b i n d i n g o f HgCI 2 t o D N A w i t h its i n d u c t i o n o f DNA repair and single s t r a n d breaks. CHO cells were e x p o s e d t o HgCl 2 for 1 h at t h e c o n c e n t r a t i o n s s h o w n in t h e figure. D N A repair activity was assessed utilizing CsCl d e n s i t y gradient s e d i m e n t a t i o n as d e s c r i b e d in t h e M e t h o d s section. Binding o f Hg t o D N A and t h e induct i o n o f single s t r a n d breaks were assessed as d e s c r i b e d in t h e M e t h o d s section.
222 HgC12 induced true single strand breaks and not alkaline labile sites or double strand breaks in the DNA. The number of agents with known capability to induce double strand breaks in the DNA is quite small and generally these agents produce both single and double strand breaks [17]. However, a number of agents are capable of producing alkaline labile sites [18,19] and it is important to determine if the single strand breaks are due to the alkaline pH required to elute DNA. Such information may yield some understanding of the interaction of an agent with DNA and may allow correlations to be made between specific DNA lesions and oncogenic activity. Interestingly, HgCl2 did not induce alkaline labile sites while preliminary data demonstrates that CaCrO4 produces alkaline dependent strand breaks. CaCrO4 [20] as well as other metal carcinogenic agents such as certain nickel compounds [21] induced DNA-protein crosslinks, while HgCl~ did not produce this lesion. Organic carcinogenic agents such as benzo[a] pyrene [22] are known to also cause the formation of DNA-protein cross-links as well. HgC12, however, produces DNA-DNA crosslinks which with time progressively increased in extent. These crosslinks are probably due to the ability of HgCl2 to interact with the bases directly [5] while the single strand breaks may result from the production of oxygen radicals by HgC12 and also by its interaction with DNA bases [3]. Such radicals have been postulated to mediate the X-ray-induced DNA damage of cells. These studies illustrate basic differences in the way metal compounds interact with DNA in intact cells. A complete understanding of the specific mode of interaction of the metal with DNA will be enhanced by studying the specific lesions produced with adequate methodology to distinguish among the possible lesions. The DNA lesions produced by HgCl2 must be considered in a different way from the DNA lesions induced by other agents. For example the single strand breaks induced by nickel compounds and CaCrO4 are repaired [20,21] while the strand breaks induced with HgC12 are not readily repaired [5]. At a low concentration of HgCl2, where strand breaks could not be detected by alkaline elution and Hg binding to DNA was also below the level of detection, DNA repair activity was elevated. Perhaps the repair activity at these low concentrations of HgCl2 was activated due to the presence of undetectable DNA lesions, but the metal concentration was not sufficient to inhibit repair. At higher concentrations of HgC12 DNA repair activity was inhibited, resulting in the detection of both strand breaks and Hg-DNA adducts. Hg is an extremely reactive metal that has a high affinity for sulfhydryl groups such as those present in proteins but the binding sites contained in DNA bases would have a considerably lower affinity. However, due to the critical function of DNA in the cell, its concentration and the fact that repair enzymes are inhibited by HgCl~ and cannot mend the DNA lesions, cell death may result directly from these genetic effects. In fact Table II shows that a 1-h exposure to 50--75 gM HgCI2 results in a high percentage of cell death and at these concentrations strand breaks and Hg binding to DNA are considerable (Fig. 8). Injury to the cell membrane has been
223 p u r p o r t e d t o be t h e basis o f Hg 2÷ c y t o t o x i c action. Although it is difficult t o precisely e x t r a p o l a t e c o n c e n t r a t i o n s o f HgC12 t h a t p r o d u c e cell memb r a n e injury to t h e levels t h a t cause D N A lesions because o f differences in c u l t u r e media, lack o f Hg u p t a k e m e a s u r e m e n t s in o t h e r systems etc., t h e levels o f Hg 2÷ t h a t p r o d u c e DNA lesions are at least as low if n o t l o w e r t h a n t h o s e t h a t injure t h e cell m e m b r a n e [ 2 3 ] . Since Hg 2~ has X-ray like effects in being able t o p r o d u c e o x y g e n radicals in cells [ 3 ] , and d e p l e t e cellular r e d u c e d g l u t a t h i o n e levels [4] t h e D N A m u s t be c o n s i d e r e d a target site o f its t o x i c action. If i n d u c t i o n o f D N A lesions and active repair o f t h e s e lesions are i m p o r t a n t for m u t a g e n i c i t y or c a r c i n o g e n i c i t y o f a chemical agent, t h e n Hg m a y be e x p e c t e d t o have weak m u t a g e n i c activity at low c o n c e n t r a t i o n s b u t at higher c o n c e n t r a t i o n s w h e r e D N A repair activity was inhibited t h e r e should be less mutagenic activity. Additionally, the D N A lesions induced b y HgC12 m a y result in miscoding during DNA replicat i o n ; h o w e v e r HgC12 has been s h o w n t o inhibit cell growth specifically in S phase [24] and t h e r e f o r e miscoding during D N A replication must o c c u r at c o n c e n t r a t i o n s o f HgC12 t h a t allow this process t o p r o c e e d in o r d e r t o achieve a m u t a g e n i c response in a surviving cell. These mechanistic findings m a y help explain the low m u t a g e n i c / c a r c i n o g e n i c activity displayed b y HgC12 in a n u m b e r o f e x p e r i m e n t a l systems [ 2 5 ] . ACK NOWLEDG EMENTS This w o r k was s u p p o r t e d by G r a n t R - 8 0 8 0 4 8 f r o m t h e United States E n v i r o n m e n t a l P r o t e c t i o n Agency, by C o n t r a c t D E - A S 0 5 - 8 1 E R 6 0 0 1 6 f r o m t h e U n i t e d States D e p a r t m e n t o f Energy and b y National Institutes o f Health G r a n t CA 2 9 5 8 1 f r o m t h e National Cancer Institute. T h e Environm e n t a l P r o t e c t i o n A g e n c y does n o t necessarily e n d o r s e any c o m m e r i c a l p r o d u c t s used in this s t u d y and t h e c o n c l u s i o n s r e p r e s e n t t h e views o f t h e a u t h o r s and do n o t r e p r e s e n t t h e opinions, policies or r e c o m m e n d a t i o n o f the E n v i r o n m e n t a l P r o t e c t i o n Agency. T h e a u t h o r s t h a n k Ms. Linda H a y g o o d for secretarial assistance and J. Daniel Heck for critically reading this m a n u s c r i p t . REFERENCES 1 M.W. Williams, J.D. Hoeschele, J.E. Turner, K.B. Jaeobson, N.T. Christie, C.L. Paton, L.H. Smith, H.R. Witschi and E.H. Lee, Chemical softness and acute metal toxicity in mice and drosophila, Toxicol. Appl. Pharmacol., 63 (1982) 461. 2 B.L. Vallee and D.D. Ulmer, Biochemical Effects of mercury, cadmium and lead, Annu. Rev. Biochem., 41 (1972) 91. 3 0 . Cantoni, S.H. Robison, R.M. Evans, N.T. Christie, D.B. Drath and M. Costa, Possible involvement of superoxide free radicals in the HgCl: induced DNA damage in CHO cells, Fed. Proc., 42 (1983) 1135. 4 0 . Cantoni, R.M. Evans and M. Costa, Similarity in the acute cytotoxic response of mammalian cells to mercury II and X-rays: DNA damage and glutathione depletion, Biochim. Biophys. Res. Commun., 108 (1982) 614.
224 5 0 . Cantoni and M. Costa, Correlations of DNA strand breakage and their repair with cell survival following acute exposure to mercury (If) and X-rays, Mol. Pharmacol., 24 (1983) 84. 6 J.D. Chapman and C.J. Gillespie, Radiation-induced events and their time scale in mammalian cells, Adv. Radiat. Biol., 9 ( 1 9 8 l ) 143. 7 O. Cantoni and M. Costa, Characterization and mechanism of DNA damage induced by chromium (VI) and mercury (II) in cultured mammalian cells, Proc. Am. Assoc. Cancer Res., 24 (1983) 74. 8 K.W. Kohn, L.C. Erickson, R.A.G. Ewig and C.A. Friedman, Fractionation of DNA from mammalian cells by alkaline elution, Biochemistry, 15 (1976) 4629. 9 K.W. Kohn, R.A.G. Ewig, L.C. Erickson and L.A. Zwelling, Measurement of strand breaks and cross-links by alkaline elution, in: E. Friedberg and P. Hanawalt (Eds.), DNA Repair: A Laboratory Manual of Research Procedures, Marcel Dekker, New York, 1981, pp. 379--401. 10 M.O. Bradley and K.W. Kohn, X-ray induced DNA double strand breaks production and repair in mammalian cells as measured by neutral filter elution, Nucleic Acids Res., 7 (1979) 793. 11 P.R. Cook and J.A. Brazell, Detection and repair of single-strand breaks in nuclear DNA, Nature, 263 (1976) 679. 12 C.A. Smith, P.K. Cooper and P.C. Hanawalt, Measurement of repair replication by equilibrium sedimentation, in: E. Friedberg and P.C. Hanawalt (Eds.), DNA Repair: A Laboratory Manual of Research Procedures, Marcel Dekker, New York, 1981, pp. 289--305. 13 M. Gross-Bellard, P. Oudet and P. Chambon, Isolation of high-molecular weight DNA from mammalian cells, Eur. J. Biochem., 36 (1973) 32. 14 N.T. Christie, O. Cantoni, R.M. Evans, R.E. Meyn and M. Costa, Use of mammalian DNA repair deficient mutants to assess the effects of toxic metal compounds on DNA, Biochem. Pharmacol. { 1984) in press. 15 D.L. Dougle, C.J. Gillespie and J.D. Chapman, DNA strand breaks, repair, and survival in x-irradiated mammalian cells, Proc. Natl. Acad. Sci. U.S.A., 73 (1976)809. 16 M.P. Abbracchio, R.M. Evans, J.D. Heck, O. Cantoni and M. Costa, The regulation of ionic uptake and cytotoxicity by specific amino acids and serum components, Biol. Trace Elem. Res., 4 {1982) 289. 17 W.E. Ross and M.O. Bradley, DNA double strand breaks in mammalian cells after exposure to intercalating agents, Biochim. Biophys. Acta, 654 (1981) 129. 18 N.R. Drinkwater and E.C. Miller, Estimation of apurinic/apyrimidinic sites and phoshotriesters in deoxyribonucleic acid treated with electrophilic carcinogens and mutagens, Biochemistry, 19 (1980) 5087. 19 D.H. Phillips and P.C. Hanawalt, Alkalisensitive sites in DNA from human cells treated with ultraviolet light, l'-acetoxysagrole or l'-acetoxyestragole, Carcinogenesis, 3 (1982) 935. 20 M.J. Tsapakos, T.H. Hampton and K.W. Wetterhahn Jennette, The carcinogen chromate induces DNA cross-links in rat liver and kidney, J. Biol. Chem., 256 (1981) 3623. 21 R.B. Ciccarelli and K.E. Wetterhahn, Nickel distribution and DNA lesions induced in rat tissues by the carcinogen nickel carbonate, Cancer Res., 42 (1982) 3544. 22 C.M. Ireland, A. Eastman and E. Bresnick, DNA-protein crosslinks induced in a hamster tracheal epithelial cell line by benzo(a)pyrene, Biochem. Biophys. Res. Commun., 109 {1982) 1291. 23 E. Walum and H. Marchner, Effects of mercuric chloride on membrane integrity of cultured cells, Toxicol. Lett., 18 (1983) 89. 24 M. Costa, O. Cantoni, M. deMars and D.E. Swartzendruber, Toxic metals produce an S-phase-specific cell cycle block, Res. Commun. Chem. Pathol. Pharmacol., 38 (1983) 405. 25 A. Leonard, P. Jacquet and R.R. Lauwerys, Mutagenicity and teratogenicty of mercury compounds, Mutat. Res., 114 {1983) 1.