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Selective effects of metal ions on RNA synthesis rates
Toxicology, 22 (1981) 9-.21 Elsevier/North-Holland Scientific Publishers Ltd.
S E L E C T I V E E F F E C T S O F M E T A L IONS ON R N A S Y N T H E S I S R A T E S *
SALIL K. NIYOGI a, ROSE P. FELDMAN a and DAVID J. HOFFMAN b
Biology Division a, Oak Ridge National Laboratory, Oak Ridge, TN 37830 and U.S. Department o f Interior b, Environmental Physiology and Toxicology Section, Patuxent Wildlife Research Center, Laurel, MD 20811 (U.S.A.) (Received June 1st, 1981 ) (Accepted July 28th, 1981)
SUMMARY
T h e effects o f 14 metal ions (chlorides) on the transcription of calf t h y m u s DNA and phage T 4 DNA with Escherichia coli R N A p o l y m e r a s e were tested. T h e s e assays were c o n d u c t e d u n d e r i m p r o v e d c o n d i t i o n s o f lower pH and in the absence o f 2 - m e r c a p t o e t h a n o l to p e r m i t greater stability o f the metal ions in solution. A m o n g the divalent metal ions tested, t h e conc e n t r a t i o n - d e p e n d e n t o r d e r o f i n h i b i t i o n o f overall t r a n s c r i p t i o n is pb 2+ ~ Zn 2+ > Cu 2+ > Be 2+ > Cd :+ > Ni 2+ > Ca 2+ :> Co :+ :> Mn 2+ > Mg 2+ > Sr 2 + and is the same with e i t h e r template. At pH 7.4 and in the absence o f 2 - m e r c a p t o e t h a n o l , considerably lower c o n c e n t r a t i o n s of several of t h e divalent metal ions are n e e d e d for inhibition o f overall t r a n s c r i p t i o n than at pH 8.1 and in the presence o f 2 - m e r c a p t o e t h a n o l . Ca :+, Mg ++, Sr ++, Zn :+, Li +, Na +, and K ÷ - c o n s i d e r e d to be n o n - m u t a g e n i c and n o n - c a r c i n o g e n i c decrease chain initiation (measured with T 4 DNA) at c o n c e n t r a t i o n s t h a t inhibit overall transcription. Pb :+, Cd 2+, Co :+, Be :+, and Mn :+ - - a l l mutagenic or carcinogenic -- stimulate chain initiation (although at widely different rates) at c o n c e n t r a t i o n s t h a t inhibit overall transcription. Cu 2+ and Ni 2 + - b o t h c a r c i n o g e n i c - stimulate initiation o n l y at very low c o n c e n t r a tions, f o l l o w e d by a progressive decrease in initiation at c o n c e n t r a t i o n s t h a t inhibit overall transcription.
INTRODUCTION
T h e ability o f metal ions to react with a variety of e l e c t r o n d o n o r sites on *Research sponsored by the Office of Health and Environmental Research, U.S. Department of Energy, under contract W-7405-eng-26 with the Union Carbide Corporation. Abbreviation: TCA, trichloroacetic acid.
9
polynucleotides [1,2 ] as well as to provide optimal conditions for the R N A polymerase reaction has received considerable attention [3--10]. Certain metals have been identified as potential environmental carcinogens through occupational exposure as well as in the laboratory [11,12]. Metal ions in this category, as well as metal mutagens, have been shown to decrease the fidelity of DNA synthesis in vitro [13]. At concentrations that inhibit overall RNA synthesis, such metal ions also stimulate R N A chain initiation in vitro, whereas metal ions not in this category inhibit chain initiation [14]. Some of these metal ions also increase misincorporation of nucleotides during RNA synthesis in vitro [15--20]. Studies o f replication and transcription are usually conducted at an optimal alkaline pH ( 7 . 8 - 8 . 1 ) and in the presence of sulfhydryl reducing agents; these conditions can cause precipitation of many of the divalent test metals, particularly at higher metal ion concentration, and thereby cause artifacts and difficulty in assessing the effective metal ion concentration (Niyogi and Feldman, unpublished observations). Accordingly, we felt it was important to reexamine the effects of previously tested metals [14], as well as some new ones, during transcription with Escherichia coli R N A polymerase at lower pH, namely, 7.4, and in the absence of 2-mercaptoethanol so that the metal ions remained in solution. Under these improved conditions (pH 7.4 is closer to the natural physiological pH), overall transcription is inhibited at much lower ( b y at least an order of magnitude) concentrations of the test metals. More importantly, we are still able to confirm the previous observations of Hoffman and Niyogi [14], namely, at metal ion concentrations that inhibit overall transcription, the same mutagenic and carcinogenic metals tested earlier cause increased initiation, whereas metal ions that are neither mutagenic nor carcinogenic cause decreased initiation. However, our results also suggest the need for caution in proposing simple in vitro tests as conclusive screening methods for mutagenicity and carcinogenicity. MATERIALS AND METHODS Unlabeled ribonucleoside triphosphates were products of P-L Biochemicals (Milwaukee, WI). [2-14C]UTP and [7-32P]ATP were purchased from Schwarz/Mann (Orangebur~, NY) and Amersham/Searle (Arlington Heights, IL), respectively. Calf t h y m u s DNA (Worthington Biochemical Corp., Freehold, NJ) was reextracted with phenol and dialyzed against 20 mM Tris--HCl (pH 7.4). Phage T4 DNA was isolated by gentle phenol extraction as described by Thomas and Abelson [21], then dialyzed against 20 mM Tris--HCl (pH 7.4} and stored at 0°C. Freezing and thawing of the T4 DNA was avoided to minimize breakage of the DN~. RNA polymerase from E. coil B was purified according to the method of Stevens [22], except that the final sucrose density gradient centrifugation step was replaced by the glycerol gradient centrifugation procedure of Burgess [23]. The preparation was more than 90% pure, as judged by
10
electrophoresis on polyacrylamide gels in the presence of sodium dodecyl sulfate. It was free of contaminating activities such as polynucleotide phosphorylase and DNA polymerase and of detectable nuclease activity, as measured b y using radioactively labeled R N A and DNA preparations as substrates for the release of acid-soluble radioactive material. The preparation did not introduce single-strand nicks in DNA, as detected b y alkaline sucrose density gradient centrifugation. Solutions of the test metal chlorides were freshly prepared before each experiment. This was necessary because prolonged storage of some of the solutions, particularly in the freezer, led to alterations in the solutions, resulting in non-reproducible results. The reaction mixture (0.2 rnl) for measuring the overall rate of R N A synthesis (reaction mixture A) contained 20 mM Tris--HCl (pH 7.4); 10 mM MgCl2; ATP, GTP, CTP, and [ 2 ) 4 C ] U T P (4000 cpm/nmol) at 0.25 mM each; 10 ~g of either calf thymus or phage T4 DNA; 5 pg of E. coli R N A polymerase holoenzyme; and varying concentrations of the test metal chloride. The reaction mixture (0.2 ml) for measuring the rate of chain initiation (reaction mixture B) contained 20 mM Tris--HC1 (pH 7.4); 10 mM MgCl2; CTP, GTP, UTP, and [7)2P]ATP (2--4 X 10 s cpm/nmol) at 0.25 mM each; 10 gg of phage T4 DNA; 5 pg of E. coil RNA polymerase holoenzyme; and varying concentrations of the test metal chloride. Each reaction was initiated b y the addition of MgCI2, and the isotope incorporation into acid-insoluble material was measured after a 10-rain incubation period at 37°C. Siliconized tubes are recommended for the assays, especially for the initiation reaction. The overall reaction was terminated by the addition of 2 ml of cold 5% trichloroacetic acid (TCA) containing 10 mM sodium pyrophosphate. After standing in ice for 10 min, the solution was filtered through a Whatman glass paper (GF/C) disc. The disc was washed extensively with the same solvent, washed again with cold ethanol, then dried under an infrared heat lamp, placed in a scintillation vial, and counted with BBOT-toluene solution (4 g BBOT/liter toluene) in a Packard Tri-Carb liquid scintillation spectrometer. The initiation reaction was terminated by the addition of 0.2 ml of 5 mM ATP containing 10 mM sodium pyrophosphate, followed immediately by the addition of 2 ml of 5% TCA containing 1 mM ATP and 10 mM sodium pyrophosphate. After standing in ice for 10 rain, the solution was filtered through a Whatman glass paper (GF/C) disc. The disc was first washed with five 2-ml portions of 5% TCA containing 1 mM ATP and 10 mM sodium pyrophosphate, then washed extensively with 5% TCA containing 10 mM sodium pyrophosphate, and finally washed with ethanol, dried, and counted as described above. RESULTS Effect o f reaction conditions on transcription efficiency Overall synthesis, as measured by incorporation of ~4C-labeled UMP, is only slightly (by 10--15%) inhibited by the new assay conditions, namely
11
lower pH (7.4) and omission o f 2 - m e r c a p t o e t h a n o l . Chain initiation, as m e a s u r e d b y i n c o r p o r a t i o n o f [7-32p]ATP, is practically u n a l t e r e d b y t h e new assay c o n d i t i o n s .
Effects of metal ion concentration on overall transcription T h e effects o f increasing c o n c e n t r a t i o n s of various metal chlorides on overall t r a n s c r i p t i o n (as r e f l e c t e d b y i n c o r p o r a t i o n o f '4C-labeled UMP) d i r e c t e d b y b o t h calf t h y m u s D N A and phage T 4 D N A are s h o w n in Figs. 1 and 2. T h e r e a c t i o n m i x t u r e s were m a i n t a i n e d at 10 mM MgC12. As s h o w n in Figs. 1 and 2, a m o n g t h e divalent metal ions tested, t h e c o n c e n t r a t i o n d e p e n d e n t o r d e r o f inhibition (based on 50% inhibition) is Pb 2~ > Z n : ' > Cu :÷ > Cd 2+ > Ni 2÷ > Ca 2. > Co 2+ > Mn 2÷, and is the same with either t e m p l a t e . Be 2÷ is m o r e i n h i b i t o r y t h a n Cd ~-÷ at low c o n c e n t r a t i o n s b u t less so at higher c o n c e n t r a t i o n s . Mn 2. stimulates overall t r a n s c r i p t i o n at low c o n c e n t r a t i o n s , reaching a m a x i m u m at 1 mM. S r : ' has very little e f f e c t on overall t r a n s c r i p t i o n ; at Sr -'÷ c o n c e n t r a t i o n s o f 10, 20, 40, 60, and 100 mM, the r e a c t i o n rates with phage T4 D N A as t e m p l a t e are 90, 80, 73, 63, and 23% o f c o n t r o l , respectively. Similar results are o b t a i n e d with calf t h y m u s DNA as t e m p l a t e . It is i m p o r t a n t to n o t e that considerably lower c o n c e n t r a t i o n s of several o f t h e divalent test metal chlorides are n e e d e d for inhibition of overall t r a n s c r i p t i o n at pH 7.4 and in t h e absence o f 2 - m e r c a p t o e t h a n o l than at pH 8.1 and in the presence o f 2 - m e r c a p t o e t h a n o l [ 1 4 ] . This fact is illustrated in Table I (derived f r o m Figs. 1 and 2 and f r o m similar curves
_100-
A Coif -]--h-imu~ DNA ~,
&
Phage T4 DNA
,, t:l\
lO
50
'(X) METAL
250 10 50 100 ON CONCN. ( ~ M )
-..
250
Fig, 1. Eft'ecL~ o f m e t a l ion c o n c e n t r a t i o n o n overall t r a n s c r i p t i o n w i t h calf t h y m u s D N A (A) and phage T4 DNA (B). Overall t r a n s c r i p t i o n was m e a s u r e d as the i n c o r p o r a t i o n o f [ ' ~ C ] U M P by using reaction m i x t u r e A, a.s d ~ c r i b e d in Materials and M e t h o d s . The c o n t r o l values, based o n t h e average o f 5 d e t e r m i n a t i o n s were 2.21 nmol and 2.80 n m o l , respectively, for calf t h y m u s DNA and phage T4 DNA. The variations b e t w e e n multiple assays were within 10%.
12
A Ccif Thymus DNA
'40-
g 'CO
B >hage - a Db.A
(\
..
£o
X
!\
~,,2-
w %.
0 c;4! .
li
!~
,::
?
a
6
~ META.
'o
-~27
....
• -~,~"
?
"........... > ~ , ~
c,:,2"
4
~
s
'~"
ICk CC?NC~, ,'r-k,1)
Fig. 2. Effects o f metal ion c o n c e n t r a t i o n o n overall t r a n s c r i p t i o n with calf t h y m u s D N A (A) a n d phage T4 D N A (B). Overall t r a n s c r i p t i o n was m e a s u r e d as t h e incorporat i o n o f [ ' ~ C ] U M P b y using r e a c t i o n m i x t u r e A, as described in Materials and M e t h o d s . T h e c o n t r o l values, based o n t h e average of 5 d e t e r m i n a t i o n s , were 2.21 n m o l a n d 2.80 n m o l , respectively, for calf t h y m u s D N A a n d phage T4 DNA. T h e variations b e t w e e n m u l t i p l e as.says were w i t h i n 1 0%.
o b t a i n e d at pH 8.1 and in the p r e s e n c e o f 2 - m e r c a p t o e t h a n o l ) with s o m e e x a m p l e s for b o t h calf t h y m u s D N A and phage T 4 D N A as t e m p l a t e . T h e need f o r m u c h higher c o n c e n t r a t i o n s o f s o m e m e t a l chlorides at p H 8.1 a n d in t h e p r e s e n c e o f 2 - m e r c a p t o e t h a n o l is caused b y a decrease in m e t a l ion c o n c e n t r a t i o n d u e t o p r e c i p i t a t i o n . This p r o b l e m is p a r t i c u l a r l y severe with Pb :~, Zn :+, Be :~, and Cu 2+. Effects o f non-mutagenic and non-carcinogenic metal ions on chain initiation T h e e f f e c t s o f increasing c o n c e n t r a t i o n s o f several m e t a l ions on initiation o f R N A s y n t h e s i s were d e t e r m i n e d by m e a s u r i n g t h e i n c o r p o r a t i o n o f [7-:'2P]ATP with phage T 4 D N A as t h e t e m p l a t e and in t h e p r e s e n c e of 10 mM MgCI2. With certain divalent m e t a l ions such as Ca 2., M~"2÷, Sr 2~, and Zn ~ , and with m o n o v a l e n t ions such as Li ÷, Na ÷ and K . . . . m e t a l s c o n s i d e r e d to be n o n - m u t a g e n i c and n o n - c a r c i n o g e n i c .... chain initiation d e c r e a s e s at m e t a l ion c o n c e n t r a t i o n s t h a t decrease overall t r a n s c r i p t i o n . T h e d a t a for Ca 2" and Zn 2+ are s h o w n in Fig. 3. Ca 2+ causes a c o n c e n t r a t i o n d e p e n d e n t d e c r e a s e in overall t r a n s c r i p t i o n t h a t is a c c o m p a n i e d b y a progressive d e c r e a s e in chain initiation. Zn 2÷ at very low c o n c e n t r a t i o n s (1--3 /~M) causes a small b u t r e p r o d u c i b l e increase in overall t r a n s c r i p t i o n , f o l l o w e d b y a decline at higher Zn 2" c o n c e n t r a t i o n s . T h e r e is a small
13
TABLE I E F F E C T S O F R E A C T I O N C O N D I T I O N S ON INHIBITION O F T R A N S C R I P T I O N BY M E T A L IONS a Metal ion
C o n c e n t r a t i o n (uM) o f metal ion for 50% i n h i b i t i o n pH 8.1, with mercaptoethanol
pH 7.4, no mercaptoethanol
With calf t h y m u s D N A as template Pb 2. Zn 2+ Cu 2+ Cd 2* Ni 2.
100 800 2000 350 2500
5 10 15 45 250
With phage T4 D N A as template Pb ~" Zn 2+ Cu 2* Cd 2+ Ni 2+
120 1200 1800 400 2000
5 ]2 20 25 1 80
a Overall t r a n s c r i p t i o n was m e a s u r e d as t h e i n c o r p o r a t i o n o f [J4C ]UMP by using r e a c t i o n m i x t u r e A, as d e s c r i b e d in Materials and M e t h o d s . N u m b e r s in c o l u m n 3 w e r e o b t a i n e d f r o m t h e curves in Figs. 1 and 2. T h o s e in c o l u m n 2 were o b t a i n e d f r o m similar curves b u t assays w e r e p e r f o r m e d at pH 8 . l in t h e p r e s e n c e o f 2 - m e r c a p t o e t h a n o l . The c o n t r o l values (based o n t h e average o f 5 d e t e r m i n a t i o n s ) u n d e r t h e latter c o n d i t i o n s are 2.36 p m o l and 3.04 p m o l , respectively, w i t h calf t h y m u s DNA and phage T4 DNA.
120] A
C(]2+
12C)- B/c ,oo-
\\
Zn ~+
+
I 0
U3
"
<~
0 -..~..
Cr ~20
~
0
,
i
4
r
0
I
~ o '
I
8 12 C(]CI2 (mM)
20 ,
1~5
0
,
I
4
r
I
r
1
8 12 ZnC! 2 (,u.M)
,
i
16
Fig. 3. E f f e c t s o f Ca 2÷ and Zn 2' c o n c e n t r a t i o n s o n R N A s y n t h e s i s with phage T4 DNA. Overall t r a n s c r i p t i o n (()) was m e a s u r e d as t h e i n c o r p o r a t i o n o f [t4C]UMP by using r e a c t i o n m i x t u r e A, whereas initiation (.') was m e a s u r e d as the i n c o r p o r a t i o n o f [7-32P]ATP by using r e a c t i o n m i x t u r e B, as described in Materials and M e t h o d s . The c o n t r o l values, based o n t h e average o f 7 determir, ations, for the i n c o r p o r a t i o n o f [1'CLUMP and [~-32P]ATP were 3.42 n m o l and 5.42 p m o l , r ~ p e c t i v e l y . Variations were as d e s c r i b e d in Figs. 1 and 2.
14
(up to 7%) stimulation of chain initiation at low concentrations of Zn :+ that decrease overall transcription. Higher concentrations of Zn :÷ inhibit chain initiation along with overall transcription. Mg :÷ (up to 40 mM) causes an increase in overall transcription {probably an ionic strength effect) that decreases at higher Mg2÷ concentrations. Chain initiation increases up to 30 mM Mg2÷ and then decreases at higher Mg :÷ concentrations. Other metal ions that are neither mutagenic nor carcinogenic, for example, Sr :÷, Li+, Na ÷, and K ÷, behaved like Ca:÷; at metal ion concentrations that inhibit overall transcription, chain initiation is also inhibited {data not shown). Effects o f mutagenic and carcinogenic metal ions on chain initiation Several divalent metal ions that have been shown to be mutagenic and carcinogenic (for known metal mutagens and carcinogens used in this study see [11], [12], and references cited in [14]) were tested for their effects on RNA synthesis rates with phage T4 DNA as the template. As shown in Fig. 4, Co 2÷, Mn :÷, and Cd 2÷ cause marked stimulation in chain initiation at concentrations that inhibit overall transcription. On a concentration b a s i s , Cd 2÷ causes the greatest stimulation in chain initiation, followed by Mn 2÷ and Co2*; Cd 2÷ produces its effect at micromolar concentrations whereas Co :÷ and Mn :÷ produce theirs at millimolar concentrations. BeClz and PbCl2, both known carcinogens [11,12], were tested for their effects on RNA chain initiation. As shown in Fig. 5, Be 2÷ and Pb :÷ cause
bOO h
6 0 0 - [~
C0 P+
Mn 2+
500- C
Cd z÷
o o ()
+o400 () 0..
400
n~
+2 /
/
i
200
/ 2+
200
100 ( / ~
,L ,'7
\ L
°"°---o
o
4 12 20 CoCl 2 (raM)
0
O~
°~'~°~o
4 12 20 MnCl 2 (mM)
20 60 100 CdCl 2 (/.,.M )
Fig. 4. Effects o f Co 2', Mn 2., and Cd 2' concentrations on R N A synthesis with phage T4 DNA. Overall transcription ( ' ) was measured as the incorporation of [ I ' C ] U M P by using reaction mixture A, whereas initiation (,~) was measured as the i n c o r p o r a t i o n of [~-32P]ATP by using reaction mixture B, as described in Materials and Methods. Control values, based on t h e average of 10 determinations, for the incorporation of [ I ' C ] U M P and [~-~2P]ATP were 3.28 nmol and 6.50 pmol, respectively. Variations were as described in Figs. 1 and 2.
15
140
[ -A-
~,
,R
Be 2+
Pb 2+
% ~2o ]
g
f~
.
/
I00-
\
%
o..o -~ 80Z ©
if_ 60<
rr
\
\
o o
E
cO 2"
,,\ \o
40
0~0.~
~ o
20 0
10~
0
i 0
~
........
50 BeCI 2 (/J.M)
T
40
.___.h 1
50
.....
0
,
-r- . . . .
~
10
'l5
l 20
r=bCI 2 ( f f M )
Fig. 5. Effects of Be ~÷ and Pb ~* concentrations on RNA synthesis with phage T4 DNA. Overall transcription (.) was measured as the incorporation of ['4C]UMP by using reaction mixture A, whereas initiation (") was measured as the incorporation of [~-~2P]ATP by using reaction mixture B, as described in Materials and Methods. The control values, based on the average of 10 determinations, for the incorporation of ['4CLUMP and [-r-~:P]ATP were 3.28 nmol and 6.50 pmol, respectively. Variations were as described in Figs. 1 and 2. o n l y a m o d e r a t e increase in initiation over a wide range of c o n c e n t r a t i o n s t h a t inhibit overall t r a n s c r i p t i o n . PbCl, at 5 pM and BeCl2 at 20 /~M cause t h e m a x i m u m s t i m u l a t i o n in chain initiation ( a b o u t 30%), while overall t r a n s c r i p t i o n is inhibited b y 60% and 55%, respectively. T h e effects o f CuCl2 and NiCl: ( a l t h o u g h Cu and Ni are k n o w n carcinogens [ 1 1 , 1 2 ] , it is u n c e r t a i n w h e t h e r their chlorides are also carcinogenic) are s h o w n in Fig. 6. Both metal ions cause, a c o n c e n t r a t i o n < t e p e n d e n t decrease in overall t r a n s c r i p t i o n , Cu 2÷ being m o r e i n h i b i t o r y than Ni 2.. B o t h p r o d u c e small initial increases in chain initiation over a n a r r o w c o n c e n t r a t i o n range, t h e e f f e c t with Cu 2+ being m o r e p r o n o u n c e d . At higher conc e n t r a t i o n s , b o t h metal ions decrease chain initiation along with overall transcription. T h e ratios o f fold increase in chain initiation to fold depression of overall synthesis with phage T 4 D N A were e x a m i n e d (Table II); these ratios were c o m p u t e d at t h e metal ion c o n c e n t r a t i o n causing t h e m a x i m u m s t i m u l a t i o n of chain initiation (Figs. 3--6). Of t h e metal m u t a g e n s and carcinogens, Pb 2+, Cd 2+, and Be 2+ have t h e greatest effect at c o n c e n t r a t i o n s o f o n l y 75 pM o r less. Mn 2+ and Co 2÷ similarly increase the ratio, but o n l y at c o n c e n t r a t i o n s 3 o r d e r s of m a g n i t u d e greater. A l t h o u g h Cu'- + and Ni: ÷ seem to cause smaller increases in initiation t h a n d o Mn 2+ and Co -'+, t h e y d o so at c o n c e n t r a t i o n s t h a t are several o r d e r s o f m a g n i t u d e lower (Table lI).
16
A
I8
Cu 2+
N, 2÷
\
~2
8
L_
o
....
6
io
¢o
o
CuGI2 (/~M)
5'0
T--
,00
r
150
2()0
N~CI2 I/.zM)
Fig. 6. E f f e c t s o f Cu 2. and Ni 2÷ c o n c e n t r a t i o n s o n R N A s y n t h e s i s w i t h phage T4 DNA. Overall t r a n s c r i p t i o n ( , ) was m e a s u r e d as t h e i n c o r p o r a t i o n o f [14C]UMP b y using r e a c t i o n m i x t u r e A, w h e r e a s initiation (,,) was m e a s u r e d as t h e i n c o r p o r a t i o n o f [~-32P]ATP by using r e a c t i o n m i x t u r e B, as described in Materials and M e t h o d s . The c o n t r o l values, based on the average o f 10 d e t e r m i n a t i o n s , for t h e i n c o r p o r a t i o n o f [~4C]UMP and [T-32P]ATP were 3.37 n m o l and 5.43 p m o l , respectively. Variations were as described in Figs. 1 and 2. T A B L E II R A T I O S O F F O L D I N C R E A S E IN CHAIN I N I T I A T I O N TO F O L D D E P R E S S I O N O F O V E R A L L S Y N T H E S I S BY M E T A L IONS WITH P H A G E T4 D N A a Metal ion
C o n c e n t r a t i o n (uM) o f metal ion at m a x i m u m s t i m u l a t i o n b o f chain initiation
Ratio o f fold increase in initiation to fold d e p r e s s i o n o f overall synthesis at m a x i m u m s t i m u l a t i o n o f chain inhalation
Cu 2+ Zn :+ Pb:" Ni 2' Be 2" Cd 2+ Mn 24 Co 2'
3 5 5 10 20 75 5 × 103 10 × l 0 s
1.6 1.4 2.9 1.3 3.3 1 1.3 9.6 14
Chain initiation o f RNA s y n t h e s i s was measured as the i n c o r p o r a t i o n o f [~-32p]ATP by using r e a c t i o n m i x t u r e B and overall synthesis as the i n c o r p o r a t i o n o f [~4C]UMP by using r e a c t i o n m i x t u r e A as described in Materials and M e t h o d s . The ratios were comp u t e d f r o m t h e curves s h o w n in Figs. 3--6. b O t h e r metals e x a m i n e d (Ca 2., Mg:*, Sr 2., Li*, Na*, K" ) decreased chain initiation at c o n c e n t r a t i o n s t h a t inhibited overall synthesis.
17
DISCUSSION Among th e metal ions tested, Mn 2÷ and Co 2÷ are the onl y metal ions {besides Mg 2~) capable o f supporting RNA synthesis with various synthetic and natural templates. In t he present studies, the different metal ions were tested in t h e presence of 10 mM MgCl2 -- an o p t i m u m c o n c e n t r a t i o n for the RNA polymerase reaction [22]. E x c e p t for Mn 2., which at low concentrations stimulates overall transcription with either calf t h y m u s DNA or phage T4 DNA as template, t he divalent metal ions inhibit overall transcription in a c o n c e n t r a t i o n - d e p e n d e n t manner. On the basis of concentrations that p r o d u ce 50% inhibition, t he order of inhibition for t he metal ions {divalent and monovalent) is Pb 2÷ > Zn 2+ > Cu 2. > Be 2+ > C d 2÷ ~> Ni 2÷ > Ca 2÷ > Co 2~ > Mn 2÷ > Mg 2÷ > Sr 2+ > Li ÷ , N a +, K ÷ and is the same with both templates. This order of inhibition may be partially explained on t he basis o f the affinities of these metal ions for nucleic acid bases compared with phosphate, namely, Pb 2÷ > Cd 2+ > Cu 2+ > Zn 2. > Mn 2. > Mg 2÷ > Li*, Na ÷, K ÷ [1,2]. D onna n equilibrium dat a are consistent with t he phosphatebinding order of Mg 2÷ > Li ÷ > Na ÷ > K ÷, which is also consistent with calculated energies o f f o r m a t i o n [ 24,25 ]. T h e strong inhibition by Zn :÷ is somewhat surprising, since E. coli RNA polymerase, and in fact all RNA polymerases studied thus far, appear to have very tightly b o u n d Zn (2 g a t o m s / e n z y m e molecule) as an integral part of th e e n z y m e [26--31]. Although the intrinsic Zn :÷ has been implicated in p r o m o t e r recognition and specific initiation of RNA synthesis [32], the detailed mechanism b y which these are achieved remains unknow n. Th e much lower (by at least an order of magnitude) concentrations of several of th e divalent test metal chlorides needed for inhibition o f overall transcription at pH 7.4 and in the absence of 2-mercaptoethanol, compared with those needed at pH 8.1 and in the presence of 2-mercaptoethanol [14], are illustrated in Table I. The results indicate that some o f the metal ions are far more t oxi c towards transcription than hitherto d e m o n s t r a t e d . T he new assay conditions result in relatively greater free concentrations of metal ions, because t he lower pH prevents precipitation of metals as h y d r o x id es and absence o f m e r c a p t o e t h a n o l prevents binding o f metal ions to sulfhydryl groups. T he improved conditions also remove the uncertainty as to the actual c o n c e n t r a t i o n of t he metal ions in solution. Phage T4 DNA was chosen for the chain initiation studies because it is a physiological t em pl a t e for E. coli RNA polymerase which transcribes only t h e " e a r l y " phage T4 genes [33]. Initiation was measured with [7-32p]ATP because it has been shown that chain initiation with E. coil RNA polymerase h o l o e n z y m e on T4 DNA occurs p r e d o m i n a n t l y with ATP rather than with GTP [8,34]. Among the clearly non-mutagenic and non-carcinogenic metals tested thus far -- Li*, Na*, K ÷, Ca:*, Mg 2÷, and Sr :÷ -- chain initiation is inhibited at metal ion concentrations that inhibit overall transcription. Zn :÷ causes only a slight initial stimulation in chain initiation at concentrations that inhibit
18
overall transcription. It is felt that Zn is a questionable carcinogen. Zinc powder does not cause tumors through intramuscular injection [35]. However, intratesticular injection o f ZnC12 has resulted in teratomas [36]. ZnC12 does not decrease the fidelity of DNA synthesis in vitro with avian myeloblastosis virus reverse transcriptase [13] but does so with rat liver DNA polymerase/3 (S. Mitra, pers. comm.). ZnCl: does not increase misincorporation of CMP during transcription of poly(dA
19
Cd 2÷ b o t h increased m i s i n c o r p o r a t i o n 10-fold at c o m p a r a b l e c o n c e n t r a t i o n s . Thus, a positive c o r r e l a t i o n b e t w e e n m e t a l - i n d u c e d m i s i n c o r p o r a t i o n during D N A replication and t h e carcinogenic p o t e n t i a l o f metal ions c o u l d not be d e m o n s t r a t e d , at least with the rat liver D N A p o l y m e r a s e . T h e increased initiation seen in t h e present studies with certain metal ions at c o n c e n t r a t i o n s t h a t decrease overall t r a n s c r i p t i o n c a n n o t be e x p l a i n e d solely o n t h e basis of increased t e r m i n a t i o n f o l l o w e d b y reinitiation, because t h e e f f e c t is n o t observed with o t h e r metal ions at c o n c e n t r a t i o n s that decrease overall t r a n s c r i p t i o n . Rather, t h e results could be explained on t h e basis o f multiple initiation at either t h e same or d i f f e r e n t sites on t h e D N A t e m p l a t e , because o f alteration d u e to t h e interaction o f the metal ion with t h e DNA. T h e i n t e r a c t i o n could be partially explained o n the basis o f t h e " h a r d - s o f t " c h a r a c t e r o f t h e metal ions [ 4 1 ] . In general, hard ions are less polarizable and p r e f e r to bind with hard a c c e p t o r s , whereas soft ions are m o r e polarizable and p r e f e r to bind with soft a c c e p t o r s . F o r e x a m p l e , a m o n g metals, Na ÷, Mg 2÷, and Ca 2÷ are classified as hard; Co 2* and Zn 2' as borderline; and Pb 2., Cd 2~, and Hg 2÷ as soft. T h e i n t e r a c t i o n o f metal ions with nucleic acids can be c h a r a c t e r i z e d a c c o r d i n g t o t h e p r e f e r e n c e o f metal ions for hard sites like p h o s p h a t e or soft sites t h a t are electron-rich, like the bases. On this basis, the affinities o f metal ions for nucleic acid bases c o m p a r e d to p h o s p h a t e are as follows: Pb 2. > Cd -~÷ > Cu 2" > Mn 2÷ Zn 2÷ > Ni 2+ - Co 2. > Mg 2~ > Ca 2. > Li " , N a * , K ' [1,2]. It is interesting t h a t metals t h a t are c o n s i d e r e d to be n o n - m u t a g e n i c or n o n - c a r c i n o g e n i c can also be classified as hard ions, whereas metals t h a t are m u t a g e n i c or carcinogenic also t e n d t o he soft. In conclusion, w h e n assessing t h e degree of p o t e n t i a l physiological impact and p o t e n t i a l t o x i c i t y o f metal ions using the present in vitro m e a s u r e m e n t s o f R N A synthesis, o n e m u s t consider at least 3 factors: (1) t h e r e q u i r e d c o n c e n t r a t i o n o f m e t a l ion to depress overall R N A synthesis; (2) w h e t h e r t h e r e is s t i m u l a t i o n (or lack o f depression) of initiation of synthesis d u r i n g d e p r e s s i o n o f overall synthesis; and (3) the ratio o f fold m a x i m u m stimulat i o n o f initiation t o the c o r r e s p o n d i n g value of fold depression o f overall synthesis at t h a t c o n c e n t r a t i o n o f metal ion. Of the metals studied and on t h e basis o f f a c t o r (1), Pb 2., Zn 2~, Cu 2., Cd 2*, Ni 2~, and Be-" would be most likely to have a physiological impact because o f t h e relatively low concent r a t i o n s n e e d e d to depress overall synthesis. However, o n the basis o f factors (2) and (3), Pb:*, Cd 2., and Be 2. w o u l d appear to have p o t e n t i a l l y the most t o x i c impact as r e f l e c t e d by the ratio of stimulation of initiation to d e p r e s s i o n o f overall synthesis. In s u p p o r t o f this c o n t e n t i o n , these 3 metals are rec.'ognized as being not o n l y mutagenic and carcinogenic but also generally t o x i c [37 ] b y o n e or m o r e r o u t e s o f e x p o s u r e [ 4 2 ] . REFERENCES
1 G.L. Eichhorn and A.Y. Shin, J. Am. Chem. Soc., 90 (1968) 7323. 2 R.M. lzatt, J.J. Christen.sen and J.H. Rytting, Chem. Rev., 71 (1971) 439.
20
3 E. Fuchs, R.L. Millette, W. Zillig and G. Walter, Eur. J. Biochem., 3 (1967) 183. 4 A.G. So, E.W. Davie, R. Epstein and A. Tissieres, Proc. Natl. Acad. Sci. U.S.A., 58 (1967) 1739. 5 U. Maitra and F. Barash, Proc. Natl. Acad. Sci. U.S.A., 64 (1969) 779. 6 J.P. Richardson, J. Mol. Biol., 49 (1970) 235. 7 H. Bremer, Cold Spring Harbor Symp. Quant. Biol., 35 (1970) 109. 8 D.J. Hoffman and S.K. Niyogi, Biochim. Biophys. Acta, 299 (1973) 588. 9 W.F. Mangel and M.J. Chamberlin, J. Biol. Chem., 249 {1974) 3002. 10 E. Domingo, R.C. Escarmis and R.C. Warner, J. Biol. Chem., 250 (1975) 2872. 11 F.W. Sunderman, Jr., in R.A. Goyer and M.A. Mehlman (Eds.), Advances in Modern Toxicology, Vol. 2, Hemisphere, Washington, D.C., 1977, p. 257. 12 F.W. Sunderman, Jr., Fed. Proc., 37 (1978)40. 13 M.A. Sirover and L.A. Loeb, Science, 194 (1976) 1434. 14 D.J. Hoffman and S.K. Niyogi, Science, 198 (1977) 513. 15 M.J. Chamberlin, R. Baldwin and P. Berg, J. Mol. Biol., 7 (1963) 334. 16 H. Bujard and C. Heidelberger, Biochemistry, 5 (1966} 3339. 17 V. Paetkau, M.B. Coulter, W.F. Flintoff and R.A. Morgan, J. Mol. Biol., 71 (1972) 293. 18 G.F. Strniste, D.A. Smith and F.N. Hayes, Biochemistry, 12 (1973) 603. 19 C.F. Springgat'e and L.A. Loeb, J. Mol. Biol., 97 (1975} 577. 20 S.K. Niyogi and R.P. Feldman, Nucleic Acids Res., 9 (1981) 2615. 21 C.A. 2+nomas, Jr., and J. Abelson, in G.L. Cantoni and D.R. Davies (Eds.), Procedures in Nucleic Acid Research, Harper and Row, New York, 1966, p. 553. 22 A. Stevens, J. Biol. Chem., 244 (1969) 425. 23 R.R. Burgess, J. Biol. Chem., 244 {1969} 6160. 24 U.P. Straus, C. Helfgott and H. Pink, J. Phys. Chem., 71 (1967) 2550. 25 D.S. Marynick and H.F. Schaefer, Proc. Natl. Acad. Sci. U.S.A., 72 (1975) 3794. 26 M.C. Scrutton, W.-W. Wu and D.A. Goldthwait, Proc. Natl. Acad. Sci. U.S.A., 68 (1971)249. 27 J.E. Coleman, Biochem. Biophys. Res. Commun., 60 (1974) 641. 28 K.H. Falchuk, B. Mazus, C. Ulpino and P. Valenzuela, Biochemistry, 15 (1976) 4468. 29 D.S. Auld, 1. Atsuya, C. Capino and P. Valenzuela, Biochem. Biophys. Res. Commun., 69 (1976) 548. 30 H. Lattke and U. Weser, FEBS Lett., 65 (1976) 288. 31 T.M. Wandzilak and R.W. Benson, Biochem. Biophys. Res. Commun., 76 (1977) 247. 32 D.C. Speckhard, F. Y.-H. Wu and C.-H. Wu, Biochemistry, 16 (1977) 5228. 33 E.N. Brody and E.P. Geidusehek, Biochemistry, 9 (1970) 1300. 34 D.J. Hoffman and S.K. Niyogi, Proe. Natl. Acad. Sci. U.S.A., 70 (1973) 574. 35 J.C. Heath, M.R. Daniel, J.T. Dingle and M. Webb, Nature, 193 (1962) 592. 36 J. Guthrie and O.A. Guthrie, Cancer Res., 34 (1974) 2612. 37 J.P.W. Gilman, Can. Cancer Conf., 6 (1966) 209. 38 F.W. Sunderman, T.J. Lau, P.F. Minghetti, R.M. Maenza, N. Becket, C. Onkelinx and P.J. Goldblatt, Proc. Am. Assoc. Cancer Res., 16 (1975) 554. 39 D.J. Beach and F.W. Sunderman, Cancer Res., 30 (1970) 1645. 40 H.R. Witsehi, Cancer Res., 32 (1972) 1686. 41 S. Ahrland, in Structure and Bonding, Vol. 5, Springer-Verlag, New York, 1968, p. 118. 42 R.B. Belisles, in L.J. Casarett and J. Doull (Eds.), Toxicology and the Basic Science of Poisons, Macmillan Publishing Co., New York, 1975, p. 454.
21
S E L E C T I V E E F F E C T S O F M E T A L IONS ON R N A S Y N T H E S I S R A T E S *
SALIL K. NIYOGI a, ROSE P. FELDMAN a and DAVID J. HOFFMAN b
Biology Division a, Oak Ridge National Laboratory, Oak Ridge, TN 37830 and U.S. Department o f Interior b, Environmental Physiology and Toxicology Section, Patuxent Wildlife Research Center, Laurel, MD 20811 (U.S.A.) (Received June 1st, 1981 ) (Accepted July 28th, 1981)
SUMMARY
T h e effects o f 14 metal ions (chlorides) on the transcription of calf t h y m u s DNA and phage T 4 DNA with Escherichia coli R N A p o l y m e r a s e were tested. T h e s e assays were c o n d u c t e d u n d e r i m p r o v e d c o n d i t i o n s o f lower pH and in the absence o f 2 - m e r c a p t o e t h a n o l to p e r m i t greater stability o f the metal ions in solution. A m o n g the divalent metal ions tested, t h e conc e n t r a t i o n - d e p e n d e n t o r d e r o f i n h i b i t i o n o f overall t r a n s c r i p t i o n is pb 2+ ~ Zn 2+ > Cu 2+ > Be 2+ > Cd :+ > Ni 2+ > Ca 2+ :> Co :+ :> Mn 2+ > Mg 2+ > Sr 2 + and is the same with e i t h e r template. At pH 7.4 and in the absence o f 2 - m e r c a p t o e t h a n o l , considerably lower c o n c e n t r a t i o n s of several of t h e divalent metal ions are n e e d e d for inhibition o f overall t r a n s c r i p t i o n than at pH 8.1 and in the presence o f 2 - m e r c a p t o e t h a n o l . Ca :+, Mg ++, Sr ++, Zn :+, Li +, Na +, and K ÷ - c o n s i d e r e d to be n o n - m u t a g e n i c and n o n - c a r c i n o g e n i c decrease chain initiation (measured with T 4 DNA) at c o n c e n t r a t i o n s t h a t inhibit overall transcription. Pb :+, Cd 2+, Co :+, Be :+, and Mn :+ - - a l l mutagenic or carcinogenic -- stimulate chain initiation (although at widely different rates) at c o n c e n t r a t i o n s t h a t inhibit overall transcription. Cu 2+ and Ni 2 + - b o t h c a r c i n o g e n i c - stimulate initiation o n l y at very low c o n c e n t r a tions, f o l l o w e d by a progressive decrease in initiation at c o n c e n t r a t i o n s t h a t inhibit overall transcription.
INTRODUCTION
T h e ability o f metal ions to react with a variety of e l e c t r o n d o n o r sites on *Research sponsored by the Office of Health and Environmental Research, U.S. Department of Energy, under contract W-7405-eng-26 with the Union Carbide Corporation. Abbreviation: TCA, trichloroacetic acid.
9
polynucleotides [1,2 ] as well as to provide optimal conditions for the R N A polymerase reaction has received considerable attention [3--10]. Certain metals have been identified as potential environmental carcinogens through occupational exposure as well as in the laboratory [11,12]. Metal ions in this category, as well as metal mutagens, have been shown to decrease the fidelity of DNA synthesis in vitro [13]. At concentrations that inhibit overall RNA synthesis, such metal ions also stimulate R N A chain initiation in vitro, whereas metal ions not in this category inhibit chain initiation [14]. Some of these metal ions also increase misincorporation of nucleotides during RNA synthesis in vitro [15--20]. Studies o f replication and transcription are usually conducted at an optimal alkaline pH ( 7 . 8 - 8 . 1 ) and in the presence of sulfhydryl reducing agents; these conditions can cause precipitation of many of the divalent test metals, particularly at higher metal ion concentration, and thereby cause artifacts and difficulty in assessing the effective metal ion concentration (Niyogi and Feldman, unpublished observations). Accordingly, we felt it was important to reexamine the effects of previously tested metals [14], as well as some new ones, during transcription with Escherichia coli R N A polymerase at lower pH, namely, 7.4, and in the absence of 2-mercaptoethanol so that the metal ions remained in solution. Under these improved conditions (pH 7.4 is closer to the natural physiological pH), overall transcription is inhibited at much lower ( b y at least an order of magnitude) concentrations of the test metals. More importantly, we are still able to confirm the previous observations of Hoffman and Niyogi [14], namely, at metal ion concentrations that inhibit overall transcription, the same mutagenic and carcinogenic metals tested earlier cause increased initiation, whereas metal ions that are neither mutagenic nor carcinogenic cause decreased initiation. However, our results also suggest the need for caution in proposing simple in vitro tests as conclusive screening methods for mutagenicity and carcinogenicity. MATERIALS AND METHODS Unlabeled ribonucleoside triphosphates were products of P-L Biochemicals (Milwaukee, WI). [2-14C]UTP and [7-32P]ATP were purchased from Schwarz/Mann (Orangebur~, NY) and Amersham/Searle (Arlington Heights, IL), respectively. Calf t h y m u s DNA (Worthington Biochemical Corp., Freehold, NJ) was reextracted with phenol and dialyzed against 20 mM Tris--HCl (pH 7.4). Phage T4 DNA was isolated by gentle phenol extraction as described by Thomas and Abelson [21], then dialyzed against 20 mM Tris--HCl (pH 7.4} and stored at 0°C. Freezing and thawing of the T4 DNA was avoided to minimize breakage of the DN~. RNA polymerase from E. coil B was purified according to the method of Stevens [22], except that the final sucrose density gradient centrifugation step was replaced by the glycerol gradient centrifugation procedure of Burgess [23]. The preparation was more than 90% pure, as judged by
10
electrophoresis on polyacrylamide gels in the presence of sodium dodecyl sulfate. It was free of contaminating activities such as polynucleotide phosphorylase and DNA polymerase and of detectable nuclease activity, as measured b y using radioactively labeled R N A and DNA preparations as substrates for the release of acid-soluble radioactive material. The preparation did not introduce single-strand nicks in DNA, as detected b y alkaline sucrose density gradient centrifugation. Solutions of the test metal chlorides were freshly prepared before each experiment. This was necessary because prolonged storage of some of the solutions, particularly in the freezer, led to alterations in the solutions, resulting in non-reproducible results. The reaction mixture (0.2 rnl) for measuring the overall rate of R N A synthesis (reaction mixture A) contained 20 mM Tris--HCl (pH 7.4); 10 mM MgCl2; ATP, GTP, CTP, and [ 2 ) 4 C ] U T P (4000 cpm/nmol) at 0.25 mM each; 10 ~g of either calf thymus or phage T4 DNA; 5 pg of E. coli R N A polymerase holoenzyme; and varying concentrations of the test metal chloride. The reaction mixture (0.2 ml) for measuring the rate of chain initiation (reaction mixture B) contained 20 mM Tris--HC1 (pH 7.4); 10 mM MgCl2; CTP, GTP, UTP, and [7)2P]ATP (2--4 X 10 s cpm/nmol) at 0.25 mM each; 10 gg of phage T4 DNA; 5 pg of E. coil RNA polymerase holoenzyme; and varying concentrations of the test metal chloride. Each reaction was initiated b y the addition of MgCI2, and the isotope incorporation into acid-insoluble material was measured after a 10-rain incubation period at 37°C. Siliconized tubes are recommended for the assays, especially for the initiation reaction. The overall reaction was terminated by the addition of 2 ml of cold 5% trichloroacetic acid (TCA) containing 10 mM sodium pyrophosphate. After standing in ice for 10 min, the solution was filtered through a Whatman glass paper (GF/C) disc. The disc was washed extensively with the same solvent, washed again with cold ethanol, then dried under an infrared heat lamp, placed in a scintillation vial, and counted with BBOT-toluene solution (4 g BBOT/liter toluene) in a Packard Tri-Carb liquid scintillation spectrometer. The initiation reaction was terminated by the addition of 0.2 ml of 5 mM ATP containing 10 mM sodium pyrophosphate, followed immediately by the addition of 2 ml of 5% TCA containing 1 mM ATP and 10 mM sodium pyrophosphate. After standing in ice for 10 rain, the solution was filtered through a Whatman glass paper (GF/C) disc. The disc was first washed with five 2-ml portions of 5% TCA containing 1 mM ATP and 10 mM sodium pyrophosphate, then washed extensively with 5% TCA containing 10 mM sodium pyrophosphate, and finally washed with ethanol, dried, and counted as described above. RESULTS Effect o f reaction conditions on transcription efficiency Overall synthesis, as measured by incorporation of ~4C-labeled UMP, is only slightly (by 10--15%) inhibited by the new assay conditions, namely
11
lower pH (7.4) and omission o f 2 - m e r c a p t o e t h a n o l . Chain initiation, as m e a s u r e d b y i n c o r p o r a t i o n o f [7-32p]ATP, is practically u n a l t e r e d b y t h e new assay c o n d i t i o n s .
Effects of metal ion concentration on overall transcription T h e effects o f increasing c o n c e n t r a t i o n s of various metal chlorides on overall t r a n s c r i p t i o n (as r e f l e c t e d b y i n c o r p o r a t i o n o f '4C-labeled UMP) d i r e c t e d b y b o t h calf t h y m u s D N A and phage T 4 D N A are s h o w n in Figs. 1 and 2. T h e r e a c t i o n m i x t u r e s were m a i n t a i n e d at 10 mM MgC12. As s h o w n in Figs. 1 and 2, a m o n g t h e divalent metal ions tested, t h e c o n c e n t r a t i o n d e p e n d e n t o r d e r o f inhibition (based on 50% inhibition) is Pb 2~ > Z n : ' > Cu :÷ > Cd 2+ > Ni 2÷ > Ca 2. > Co 2+ > Mn 2÷, and is the same with either t e m p l a t e . Be 2÷ is m o r e i n h i b i t o r y t h a n Cd ~-÷ at low c o n c e n t r a t i o n s b u t less so at higher c o n c e n t r a t i o n s . Mn 2. stimulates overall t r a n s c r i p t i o n at low c o n c e n t r a t i o n s , reaching a m a x i m u m at 1 mM. S r : ' has very little e f f e c t on overall t r a n s c r i p t i o n ; at Sr -'÷ c o n c e n t r a t i o n s o f 10, 20, 40, 60, and 100 mM, the r e a c t i o n rates with phage T4 D N A as t e m p l a t e are 90, 80, 73, 63, and 23% o f c o n t r o l , respectively. Similar results are o b t a i n e d with calf t h y m u s DNA as t e m p l a t e . It is i m p o r t a n t to n o t e that considerably lower c o n c e n t r a t i o n s of several o f t h e divalent test metal chlorides are n e e d e d for inhibition of overall t r a n s c r i p t i o n at pH 7.4 and in t h e absence o f 2 - m e r c a p t o e t h a n o l than at pH 8.1 and in the presence o f 2 - m e r c a p t o e t h a n o l [ 1 4 ] . This fact is illustrated in Table I (derived f r o m Figs. 1 and 2 and f r o m similar curves
_100-
A Coif -]--h-imu~ DNA ~,
&
Phage T4 DNA
,, t:l\
lO
50
'(X) METAL
250 10 50 100 ON CONCN. ( ~ M )
-..
250
Fig, 1. Eft'ecL~ o f m e t a l ion c o n c e n t r a t i o n o n overall t r a n s c r i p t i o n w i t h calf t h y m u s D N A (A) and phage T4 DNA (B). Overall t r a n s c r i p t i o n was m e a s u r e d as the i n c o r p o r a t i o n o f [ ' ~ C ] U M P by using reaction m i x t u r e A, a.s d ~ c r i b e d in Materials and M e t h o d s . The c o n t r o l values, based o n t h e average o f 5 d e t e r m i n a t i o n s were 2.21 nmol and 2.80 n m o l , respectively, for calf t h y m u s DNA and phage T4 DNA. The variations b e t w e e n multiple assays were within 10%.
12
A Ccif Thymus DNA
'40-
g 'CO
B >hage - a Db.A
(\
..
£o
X
!\
~,,2-
w %.
0 c;4! .
li
!~
,::
?
a
6
~ META.
'o
-~27
....
• -~,~"
?
"........... > ~ , ~
c,:,2"
4
~
s
'~"
ICk CC?NC~, ,'r-k,1)
Fig. 2. Effects o f metal ion c o n c e n t r a t i o n o n overall t r a n s c r i p t i o n with calf t h y m u s D N A (A) a n d phage T4 D N A (B). Overall t r a n s c r i p t i o n was m e a s u r e d as t h e incorporat i o n o f [ ' ~ C ] U M P b y using r e a c t i o n m i x t u r e A, as described in Materials and M e t h o d s . T h e c o n t r o l values, based o n t h e average of 5 d e t e r m i n a t i o n s , were 2.21 n m o l a n d 2.80 n m o l , respectively, for calf t h y m u s D N A a n d phage T4 DNA. T h e variations b e t w e e n m u l t i p l e as.says were w i t h i n 1 0%.
o b t a i n e d at pH 8.1 and in the p r e s e n c e o f 2 - m e r c a p t o e t h a n o l ) with s o m e e x a m p l e s for b o t h calf t h y m u s D N A and phage T 4 D N A as t e m p l a t e . T h e need f o r m u c h higher c o n c e n t r a t i o n s o f s o m e m e t a l chlorides at p H 8.1 a n d in t h e p r e s e n c e o f 2 - m e r c a p t o e t h a n o l is caused b y a decrease in m e t a l ion c o n c e n t r a t i o n d u e t o p r e c i p i t a t i o n . This p r o b l e m is p a r t i c u l a r l y severe with Pb :~, Zn :+, Be :~, and Cu 2+. Effects o f non-mutagenic and non-carcinogenic metal ions on chain initiation T h e e f f e c t s o f increasing c o n c e n t r a t i o n s o f several m e t a l ions on initiation o f R N A s y n t h e s i s were d e t e r m i n e d by m e a s u r i n g t h e i n c o r p o r a t i o n o f [7-:'2P]ATP with phage T 4 D N A as t h e t e m p l a t e and in t h e p r e s e n c e of 10 mM MgCI2. With certain divalent m e t a l ions such as Ca 2., M~"2÷, Sr 2~, and Zn ~ , and with m o n o v a l e n t ions such as Li ÷, Na ÷ and K . . . . m e t a l s c o n s i d e r e d to be n o n - m u t a g e n i c and n o n - c a r c i n o g e n i c .... chain initiation d e c r e a s e s at m e t a l ion c o n c e n t r a t i o n s t h a t decrease overall t r a n s c r i p t i o n . T h e d a t a for Ca 2" and Zn 2+ are s h o w n in Fig. 3. Ca 2+ causes a c o n c e n t r a t i o n d e p e n d e n t d e c r e a s e in overall t r a n s c r i p t i o n t h a t is a c c o m p a n i e d b y a progressive d e c r e a s e in chain initiation. Zn 2÷ at very low c o n c e n t r a t i o n s (1--3 /~M) causes a small b u t r e p r o d u c i b l e increase in overall t r a n s c r i p t i o n , f o l l o w e d b y a decline at higher Zn 2" c o n c e n t r a t i o n s . T h e r e is a small
13
TABLE I E F F E C T S O F R E A C T I O N C O N D I T I O N S ON INHIBITION O F T R A N S C R I P T I O N BY M E T A L IONS a Metal ion
C o n c e n t r a t i o n (uM) o f metal ion for 50% i n h i b i t i o n pH 8.1, with mercaptoethanol
pH 7.4, no mercaptoethanol
With calf t h y m u s D N A as template Pb 2. Zn 2+ Cu 2+ Cd 2* Ni 2.
100 800 2000 350 2500
5 10 15 45 250
With phage T4 D N A as template Pb ~" Zn 2+ Cu 2* Cd 2+ Ni 2+
120 1200 1800 400 2000
5 ]2 20 25 1 80
a Overall t r a n s c r i p t i o n was m e a s u r e d as t h e i n c o r p o r a t i o n o f [J4C ]UMP by using r e a c t i o n m i x t u r e A, as d e s c r i b e d in Materials and M e t h o d s . N u m b e r s in c o l u m n 3 w e r e o b t a i n e d f r o m t h e curves in Figs. 1 and 2. T h o s e in c o l u m n 2 were o b t a i n e d f r o m similar curves b u t assays w e r e p e r f o r m e d at pH 8 . l in t h e p r e s e n c e o f 2 - m e r c a p t o e t h a n o l . The c o n t r o l values (based o n t h e average o f 5 d e t e r m i n a t i o n s ) u n d e r t h e latter c o n d i t i o n s are 2.36 p m o l and 3.04 p m o l , respectively, w i t h calf t h y m u s DNA and phage T4 DNA.
120] A
C(]2+
12C)- B/c ,oo-
\\
Zn ~+
+
I 0
U3
"
<~
0 -..~..
Cr ~20
~
0
,
i
4
r
0
I
~ o '
I
8 12 C(]CI2 (mM)
20 ,
1~5
0
,
I
4
r
I
r
1
8 12 ZnC! 2 (,u.M)
,
i
16
Fig. 3. E f f e c t s o f Ca 2÷ and Zn 2' c o n c e n t r a t i o n s o n R N A s y n t h e s i s with phage T4 DNA. Overall t r a n s c r i p t i o n (()) was m e a s u r e d as t h e i n c o r p o r a t i o n o f [t4C]UMP by using r e a c t i o n m i x t u r e A, whereas initiation (.') was m e a s u r e d as the i n c o r p o r a t i o n o f [7-32P]ATP by using r e a c t i o n m i x t u r e B, as described in Materials and M e t h o d s . The c o n t r o l values, based o n t h e average o f 7 determir, ations, for the i n c o r p o r a t i o n o f [1'CLUMP and [~-32P]ATP were 3.42 n m o l and 5.42 p m o l , r ~ p e c t i v e l y . Variations were as d e s c r i b e d in Figs. 1 and 2.
14
(up to 7%) stimulation of chain initiation at low concentrations of Zn :+ that decrease overall transcription. Higher concentrations of Zn :÷ inhibit chain initiation along with overall transcription. Mg :÷ (up to 40 mM) causes an increase in overall transcription {probably an ionic strength effect) that decreases at higher Mg2÷ concentrations. Chain initiation increases up to 30 mM Mg2÷ and then decreases at higher Mg :÷ concentrations. Other metal ions that are neither mutagenic nor carcinogenic, for example, Sr :÷, Li+, Na ÷, and K ÷, behaved like Ca:÷; at metal ion concentrations that inhibit overall transcription, chain initiation is also inhibited {data not shown). Effects o f mutagenic and carcinogenic metal ions on chain initiation Several divalent metal ions that have been shown to be mutagenic and carcinogenic (for known metal mutagens and carcinogens used in this study see [11], [12], and references cited in [14]) were tested for their effects on RNA synthesis rates with phage T4 DNA as the template. As shown in Fig. 4, Co 2÷, Mn :÷, and Cd 2÷ cause marked stimulation in chain initiation at concentrations that inhibit overall transcription. On a concentration b a s i s , Cd 2÷ causes the greatest stimulation in chain initiation, followed by Mn 2÷ and Co2*; Cd 2÷ produces its effect at micromolar concentrations whereas Co :÷ and Mn :÷ produce theirs at millimolar concentrations. BeClz and PbCl2, both known carcinogens [11,12], were tested for their effects on RNA chain initiation. As shown in Fig. 5, Be 2÷ and Pb :÷ cause
bOO h
6 0 0 - [~
C0 P+
Mn 2+
500- C
Cd z÷
o o ()
+o400 () 0..
400
n~
+2 /
/
i
200
/ 2+
200
100 ( / ~
,L ,'7
\ L
°"°---o
o
4 12 20 CoCl 2 (raM)
0
O~
°~'~°~o
4 12 20 MnCl 2 (mM)
20 60 100 CdCl 2 (/.,.M )
Fig. 4. Effects o f Co 2', Mn 2., and Cd 2' concentrations on R N A synthesis with phage T4 DNA. Overall transcription ( ' ) was measured as the incorporation of [ I ' C ] U M P by using reaction mixture A, whereas initiation (,~) was measured as the i n c o r p o r a t i o n of [~-32P]ATP by using reaction mixture B, as described in Materials and Methods. Control values, based on t h e average of 10 determinations, for the incorporation of [ I ' C ] U M P and [~-~2P]ATP were 3.28 nmol and 6.50 pmol, respectively. Variations were as described in Figs. 1 and 2.
15
140
[ -A-
~,
,R
Be 2+
Pb 2+
% ~2o ]
g
f~
.
/
I00-
\
%
o..o -~ 80Z ©
if_ 60<
rr
\
\
o o
E
cO 2"
,,\ \o
40
0~0.~
~ o
20 0
10~
0
i 0
~
........
50 BeCI 2 (/J.M)
T
40
.___.h 1
50
.....
0
,
-r- . . . .
~
10
'l5
l 20
r=bCI 2 ( f f M )
Fig. 5. Effects of Be ~÷ and Pb ~* concentrations on RNA synthesis with phage T4 DNA. Overall transcription (.) was measured as the incorporation of ['4C]UMP by using reaction mixture A, whereas initiation (") was measured as the incorporation of [~-~2P]ATP by using reaction mixture B, as described in Materials and Methods. The control values, based on the average of 10 determinations, for the incorporation of ['4CLUMP and [-r-~:P]ATP were 3.28 nmol and 6.50 pmol, respectively. Variations were as described in Figs. 1 and 2. o n l y a m o d e r a t e increase in initiation over a wide range of c o n c e n t r a t i o n s t h a t inhibit overall t r a n s c r i p t i o n . PbCl, at 5 pM and BeCl2 at 20 /~M cause t h e m a x i m u m s t i m u l a t i o n in chain initiation ( a b o u t 30%), while overall t r a n s c r i p t i o n is inhibited b y 60% and 55%, respectively. T h e effects o f CuCl2 and NiCl: ( a l t h o u g h Cu and Ni are k n o w n carcinogens [ 1 1 , 1 2 ] , it is u n c e r t a i n w h e t h e r their chlorides are also carcinogenic) are s h o w n in Fig. 6. Both metal ions cause, a c o n c e n t r a t i o n < t e p e n d e n t decrease in overall t r a n s c r i p t i o n , Cu 2÷ being m o r e i n h i b i t o r y than Ni 2.. B o t h p r o d u c e small initial increases in chain initiation over a n a r r o w c o n c e n t r a t i o n range, t h e e f f e c t with Cu 2+ being m o r e p r o n o u n c e d . At higher conc e n t r a t i o n s , b o t h metal ions decrease chain initiation along with overall transcription. T h e ratios o f fold increase in chain initiation to fold depression of overall synthesis with phage T 4 D N A were e x a m i n e d (Table II); these ratios were c o m p u t e d at t h e metal ion c o n c e n t r a t i o n causing t h e m a x i m u m s t i m u l a t i o n of chain initiation (Figs. 3--6). Of t h e metal m u t a g e n s and carcinogens, Pb 2+, Cd 2+, and Be 2+ have t h e greatest effect at c o n c e n t r a t i o n s o f o n l y 75 pM o r less. Mn 2+ and Co 2÷ similarly increase the ratio, but o n l y at c o n c e n t r a t i o n s 3 o r d e r s of m a g n i t u d e greater. A l t h o u g h Cu'- + and Ni: ÷ seem to cause smaller increases in initiation t h a n d o Mn 2+ and Co -'+, t h e y d o so at c o n c e n t r a t i o n s t h a t are several o r d e r s o f m a g n i t u d e lower (Table lI).
16
A
I8
Cu 2+
N, 2÷
\
~2
8
L_
o
....
6
io
¢o
o
CuGI2 (/~M)
5'0
T--
,00
r
150
2()0
N~CI2 I/.zM)
Fig. 6. E f f e c t s o f Cu 2. and Ni 2÷ c o n c e n t r a t i o n s o n R N A s y n t h e s i s w i t h phage T4 DNA. Overall t r a n s c r i p t i o n ( , ) was m e a s u r e d as t h e i n c o r p o r a t i o n o f [14C]UMP b y using r e a c t i o n m i x t u r e A, w h e r e a s initiation (,,) was m e a s u r e d as t h e i n c o r p o r a t i o n o f [~-32P]ATP by using r e a c t i o n m i x t u r e B, as described in Materials and M e t h o d s . The c o n t r o l values, based on the average o f 10 d e t e r m i n a t i o n s , for t h e i n c o r p o r a t i o n o f [~4C]UMP and [T-32P]ATP were 3.37 n m o l and 5.43 p m o l , respectively. Variations were as described in Figs. 1 and 2. T A B L E II R A T I O S O F F O L D I N C R E A S E IN CHAIN I N I T I A T I O N TO F O L D D E P R E S S I O N O F O V E R A L L S Y N T H E S I S BY M E T A L IONS WITH P H A G E T4 D N A a Metal ion
C o n c e n t r a t i o n (uM) o f metal ion at m a x i m u m s t i m u l a t i o n b o f chain initiation
Ratio o f fold increase in initiation to fold d e p r e s s i o n o f overall synthesis at m a x i m u m s t i m u l a t i o n o f chain inhalation
Cu 2+ Zn :+ Pb:" Ni 2' Be 2" Cd 2+ Mn 24 Co 2'
3 5 5 10 20 75 5 × 103 10 × l 0 s
1.6 1.4 2.9 1.3 3.3 1 1.3 9.6 14
Chain initiation o f RNA s y n t h e s i s was measured as the i n c o r p o r a t i o n o f [~-32p]ATP by using r e a c t i o n m i x t u r e B and overall synthesis as the i n c o r p o r a t i o n o f [~4C]UMP by using r e a c t i o n m i x t u r e A as described in Materials and M e t h o d s . The ratios were comp u t e d f r o m t h e curves s h o w n in Figs. 3--6. b O t h e r metals e x a m i n e d (Ca 2., Mg:*, Sr 2., Li*, Na*, K" ) decreased chain initiation at c o n c e n t r a t i o n s t h a t inhibited overall synthesis.
17
DISCUSSION Among th e metal ions tested, Mn 2÷ and Co 2÷ are the onl y metal ions {besides Mg 2~) capable o f supporting RNA synthesis with various synthetic and natural templates. In t he present studies, the different metal ions were tested in t h e presence of 10 mM MgCl2 -- an o p t i m u m c o n c e n t r a t i o n for the RNA polymerase reaction [22]. E x c e p t for Mn 2., which at low concentrations stimulates overall transcription with either calf t h y m u s DNA or phage T4 DNA as template, t he divalent metal ions inhibit overall transcription in a c o n c e n t r a t i o n - d e p e n d e n t manner. On the basis of concentrations that p r o d u ce 50% inhibition, t he order of inhibition for t he metal ions {divalent and monovalent) is Pb 2÷ > Zn 2+ > Cu 2. > Be 2+ > C d 2÷ ~> Ni 2÷ > Ca 2÷ > Co 2~ > Mn 2÷ > Mg 2÷ > Sr 2+ > Li ÷ , N a +, K ÷ and is the same with both templates. This order of inhibition may be partially explained on t he basis o f the affinities of these metal ions for nucleic acid bases compared with phosphate, namely, Pb 2÷ > Cd 2+ > Cu 2+ > Zn 2. > Mn 2. > Mg 2÷ > Li*, Na ÷, K ÷ [1,2]. D onna n equilibrium dat a are consistent with t he phosphatebinding order of Mg 2÷ > Li ÷ > Na ÷ > K ÷, which is also consistent with calculated energies o f f o r m a t i o n [ 24,25 ]. T h e strong inhibition by Zn :÷ is somewhat surprising, since E. coli RNA polymerase, and in fact all RNA polymerases studied thus far, appear to have very tightly b o u n d Zn (2 g a t o m s / e n z y m e molecule) as an integral part of th e e n z y m e [26--31]. Although the intrinsic Zn :÷ has been implicated in p r o m o t e r recognition and specific initiation of RNA synthesis [32], the detailed mechanism b y which these are achieved remains unknow n. Th e much lower (by at least an order of magnitude) concentrations of several of th e divalent test metal chlorides needed for inhibition o f overall transcription at pH 7.4 and in the absence of 2-mercaptoethanol, compared with those needed at pH 8.1 and in the presence of 2-mercaptoethanol [14], are illustrated in Table I. The results indicate that some o f the metal ions are far more t oxi c towards transcription than hitherto d e m o n s t r a t e d . T he new assay conditions result in relatively greater free concentrations of metal ions, because t he lower pH prevents precipitation of metals as h y d r o x id es and absence o f m e r c a p t o e t h a n o l prevents binding o f metal ions to sulfhydryl groups. T he improved conditions also remove the uncertainty as to the actual c o n c e n t r a t i o n of t he metal ions in solution. Phage T4 DNA was chosen for the chain initiation studies because it is a physiological t em pl a t e for E. coli RNA polymerase which transcribes only t h e " e a r l y " phage T4 genes [33]. Initiation was measured with [7-32p]ATP because it has been shown that chain initiation with E. coil RNA polymerase h o l o e n z y m e on T4 DNA occurs p r e d o m i n a n t l y with ATP rather than with GTP [8,34]. Among the clearly non-mutagenic and non-carcinogenic metals tested thus far -- Li*, Na*, K ÷, Ca:*, Mg 2÷, and Sr :÷ -- chain initiation is inhibited at metal ion concentrations that inhibit overall transcription. Zn :÷ causes only a slight initial stimulation in chain initiation at concentrations that inhibit
18
overall transcription. It is felt that Zn is a questionable carcinogen. Zinc powder does not cause tumors through intramuscular injection [35]. However, intratesticular injection o f ZnC12 has resulted in teratomas [36]. ZnC12 does not decrease the fidelity of DNA synthesis in vitro with avian myeloblastosis virus reverse transcriptase [13] but does so with rat liver DNA polymerase/3 (S. Mitra, pers. comm.). ZnCl: does not increase misincorporation of CMP during transcription of poly(dA
19
Cd 2÷ b o t h increased m i s i n c o r p o r a t i o n 10-fold at c o m p a r a b l e c o n c e n t r a t i o n s . Thus, a positive c o r r e l a t i o n b e t w e e n m e t a l - i n d u c e d m i s i n c o r p o r a t i o n during D N A replication and t h e carcinogenic p o t e n t i a l o f metal ions c o u l d not be d e m o n s t r a t e d , at least with the rat liver D N A p o l y m e r a s e . T h e increased initiation seen in t h e present studies with certain metal ions at c o n c e n t r a t i o n s t h a t decrease overall t r a n s c r i p t i o n c a n n o t be e x p l a i n e d solely o n t h e basis of increased t e r m i n a t i o n f o l l o w e d b y reinitiation, because t h e e f f e c t is n o t observed with o t h e r metal ions at c o n c e n t r a t i o n s that decrease overall t r a n s c r i p t i o n . Rather, t h e results could be explained on t h e basis o f multiple initiation at either t h e same or d i f f e r e n t sites on t h e D N A t e m p l a t e , because o f alteration d u e to t h e interaction o f the metal ion with t h e DNA. T h e i n t e r a c t i o n could be partially explained o n the basis o f t h e " h a r d - s o f t " c h a r a c t e r o f t h e metal ions [ 4 1 ] . In general, hard ions are less polarizable and p r e f e r to bind with hard a c c e p t o r s , whereas soft ions are m o r e polarizable and p r e f e r to bind with soft a c c e p t o r s . F o r e x a m p l e , a m o n g metals, Na ÷, Mg 2÷, and Ca 2÷ are classified as hard; Co 2* and Zn 2' as borderline; and Pb 2., Cd 2~, and Hg 2÷ as soft. T h e i n t e r a c t i o n o f metal ions with nucleic acids can be c h a r a c t e r i z e d a c c o r d i n g t o t h e p r e f e r e n c e o f metal ions for hard sites like p h o s p h a t e or soft sites t h a t are electron-rich, like the bases. On this basis, the affinities o f metal ions for nucleic acid bases c o m p a r e d to p h o s p h a t e are as follows: Pb 2. > Cd -~÷ > Cu 2" > Mn 2÷ Zn 2÷ > Ni 2+ - Co 2. > Mg 2~ > Ca 2. > Li " , N a * , K ' [1,2]. It is interesting t h a t metals t h a t are c o n s i d e r e d to be n o n - m u t a g e n i c or n o n - c a r c i n o g e n i c can also be classified as hard ions, whereas metals t h a t are m u t a g e n i c or carcinogenic also t e n d t o he soft. In conclusion, w h e n assessing t h e degree of p o t e n t i a l physiological impact and p o t e n t i a l t o x i c i t y o f metal ions using the present in vitro m e a s u r e m e n t s o f R N A synthesis, o n e m u s t consider at least 3 factors: (1) t h e r e q u i r e d c o n c e n t r a t i o n o f m e t a l ion to depress overall R N A synthesis; (2) w h e t h e r t h e r e is s t i m u l a t i o n (or lack o f depression) of initiation of synthesis d u r i n g d e p r e s s i o n o f overall synthesis; and (3) the ratio o f fold m a x i m u m stimulat i o n o f initiation t o the c o r r e s p o n d i n g value of fold depression o f overall synthesis at t h a t c o n c e n t r a t i o n o f metal ion. Of the metals studied and on t h e basis o f f a c t o r (1), Pb 2., Zn 2~, Cu 2., Cd 2*, Ni 2~, and Be-" would be most likely to have a physiological impact because o f t h e relatively low concent r a t i o n s n e e d e d to depress overall synthesis. However, o n the basis o f factors (2) and (3), Pb:*, Cd 2., and Be 2. w o u l d appear to have p o t e n t i a l l y the most t o x i c impact as r e f l e c t e d by the ratio of stimulation of initiation to d e p r e s s i o n o f overall synthesis. In s u p p o r t o f this c o n t e n t i o n , these 3 metals are rec.'ognized as being not o n l y mutagenic and carcinogenic but also generally t o x i c [37 ] b y o n e or m o r e r o u t e s o f e x p o s u r e [ 4 2 ] . REFERENCES
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