Nick translation studies on DNA strand breaks in pBR322 plasmid induced by different chromium species

295

TXLO2170

Nick translation studies on DNA strand breaks in pBR322 plasmid induced by different chromium species

T. Wolf, H.M. Bolt and H. Ottenwiilder Institut,fiir

ArbPitspl~~siologie,

(Received

29 November

(Revision

received

(Accepted

Ke,t, ilorrl.v: Chromate;

Dortmund,

Dortmund

(F. R.G)

1988)

I3 February

14 February

Superoxide

Universitiit

1989)

1989) Hydrogen

peroxide;

Plasmid pBR322;

DNA gel electrophoresis;

Nick translation;

dismutase

SUMMARY Single strand

breaks are DNA defects caused by various chemicals.

by chromium(V1) were examined. detected

during

the reduction

Using DNA agarose

only when chromium(V1)

by an excess of glutathione double-stranded had occurred

plasmid

chromium

was reduced

complexes

and a nick translation

by hydrogen in the DNA

No strand

of superoxide

The reduction

agarose

gel electrophoresis

may be a relevant

assay, indicating

breaks could be detected

dismutase.

or hydrogen

assay, strand

peroxide.

DNA and in the nick translation

under these conditions.

breaks induced in vitro

DNA strand

species with glutathione

gel electrophoresis

led to no alteration

pBR322

gen peroxide after the addition

of this chromium

This indicates

during

were

of chromium(V1) pattern

that no strand the reduction

that hydroxyl

reactive species involved in chromate

peroxide

breaks

radicals

of the breaks

by hydro-

from peroxo-

genotoxicity.

INTRODUCTION

Epidemiological

studies have given evidence

of respiratory

tract cancer in human

subjects occupationally exposed to chromates [ 1, 21. Chromium(V1) compounds exert mutagenic effects in most cellular mutagenicity test systems, whereas Cr(III) compounds do not [3, 41. This difference reflects the selective permeability of cell membranes to Cr(VI). Cr(V1) is actively transported by

Address

for correspondence:

strasse 67, D-4600 Dortmund,

037X-4274/89/$3,50

T. Wolf, Institut

fur Arbeitsphysiologie,

Universitlt

F.R.G.

@ 1989 Elsevier Science Publishers

B.V. (Biomedical

Division)

Dortmund,

Ardey-

an anion

carrier.

and SO4 :H,PO;I tntraceltutarty. 3s

microsomes

which is responsible transport

C‘r(VI) is reduced (containing

for both HCQ

in other somatic

/Cl

exchange

to Cr(tI1) by diff‘erent subcettutar

cytochrome

in erythroq

te\

cells [S 71.

P-450) or cytosot

(containing

fractions. cysteinc

5~1~11

and

gtutathionc) [X IO]. Cr(II1) induces DNA DNA cross-links during incubation with isolated DNA [I I]. If nuclei arc used. DNA--protein cross-links are observed [I?]. When Cr(Vt) is added to intact cells, an increase in chromium-induced DNA strand breaks ib observed along with increasing glutathione (GSH) tcvets in these cells [ 131.On the other hand. Cr(V1) does not react with I)NA until reduction to (‘r(IIl) by reducing agents occurs [141. IJsing agarose get etectrophoresis and nick translation [ 15. 161. this study examines the ability of Cr(V1) to induce strand breaks in DNA in vitro, and the influence of reducing Cr(Vt) by GSH or hydrogen peroxide (H?Oz) on this efrect. Nick translation is ;I method ~~suatty implemented for radioactive labeling of DNA. In microbiological research, DNase I is used to cause single strand breaks (nicks).

MATERIALS

AND METHODS

DNA-potymerase I (Kornberg potymerase). 5000 U/ml, DNase I from bovine pancreas, dCTP. dATP, dGTP. dTTP, plasmid pBR322 DNA and superoxide dismL!tase. 500 U/ml. were obtained from Boehringer. Mannheim. The deoxyadenosinee5-[r-%]thiophosphate, spec. act. 650 Ciimmot, and deoxycytidine-5-[a-‘“Slthiophosphate, spec. act. 1200 Ci/mmot. were obtained from Amersham. Braunschweig.

The solution of potassium pH 7.6, that of Cr( III) nitrate ione solution

(Sigma,

Munich)

dichromate (Fluka, (Aldrich. Steinheim) was 0.1 mot/l,

Neu-Ulm) used was 0. I mmot; I. was 0. I mmol; I. pH 3.2. Gtutath-

pH 7.6. Hydrogen

peroxide

was 30$

(Baker, Gross-Gerau).

50 mmot/t potassium phosphate, 0.25 mmot/t glycerol. 5 mmol/l MgC12. pH 7.2.

dithiothreitol

(Sigma,

Munich),

507;

The etectrophoresis buffer contained 89 mmoI/t Tris, X9 mmot/t H3B03 and 2.5 mmolll EDTA. pH 8.0. Agarose gets contained 0.8% agarose and 0.5 pg/mt ethidium

297

bromide

(ethidium

Electrophoresis Simple

bromide

was performed

was not used in electrophoresis for 3 h at a constant

of radioactive

DNA).

voltage (80 V).

prepurution

0.5 jig pBR322

for the gel electrophoresis

(ethidium

bromide

staining)

and I .O pg

pBR322 for the nick translation assay were incubated for I h at 37°C in a total volume of 20 ~1 with: (a) 100 pmol Cr(III); (b) 100 pmol Cr(V1); (c) 100 pmol Cr(V1) plus 200 nmol GSH; (dl) 100 pmol Cr(V1) plus 9.8 pmol HZ&; (d2) 100 pmol Cr(V1) plus 980 nmol H202; (el) 9.8 pmol H202; (e2) 980 nmol H202; (f) 100 pmol Cr(V1) plus 9.8 pmol H102 plus 1 pg superoxide dismutase; (g) like dl, but DlO instead of H20; (h) 100 pmol Cr(II1) plus 9.8 pmol H202; (j) 200 nmol GSH plus 9.8 pmol HzOz. Control incubations contained 20 ~1 aqueous plasmid DNA. After incubation, the samples were mixed with gel loading buffer (0.25% bromophenol blue, 0.25% xylene cyanol, 15% Ficoll400) and loaded into the slots of a submerged agarose gel. After incubation, the samples for nick translation were discontinuously dialysed against water for 12 h in equilibrium dialysis cells by changing the water once per hour. After dialysis, DNA was precipitated by addition of 100 ,LL~ 5 M NaCl and two volumes of ice-cold ethanol. Precipitation was completed by cooling for I h at -80 C. The DNA was sedimented by centrifugation for 1 h at 16000 x x. The pellets were washed twice with 70% ethanol and dried at room temperature in a stream of N,. The dried DNA samples were dissolved overnight (4°C) by adding 20 ~1 nick translation buffer. Nick

trunslution

To each were added phosphate adding 25

as.wy

DNA sample, 400 pmol of non-radioactive dCTP, dATP, dGTP and TTP in addition to 5 @Zi of either radioactive deoxyadenosinee5-[r-35S]thiotrior deoxycytidine-5-[a-35S]thiotriphosphate. The reaction was started by U of DNA-polymerase I and incubated for I h at 14°C. Nick translation

was terminated by addition of 2 ~1 0.5 M EDTA. Non-incorporated radioactive nucleotides were removed by (self-made) spin columns [ 13, 141. The radioactive DNA was separated by agarose gel electrophoresis (for conditions. see above). After electrophoresis, the gels were dried and covered with an X-ray film (CEA. Wicor-xRP,

Blue sensitive)

for 18 h at - 80°C.

RESULTS

When plasmid pBR322 DNA was incubated with chromium of different oxidation states, strand breaks could only be detected in incubations containing Cr(V1) and HzO1. Figs. I and 2 show the electrophoretic patterns of incubated plasmid pBR322 samples in ethidium-bromide-stained agarose gels. It is evident from lane dl (Fig. I) and lanes d2 and g (Fig. 2) that only DNA, incubated according to incubation

el

dl

Fig. I. Ethidium-bromide-st;lined

b

c

agarose

a

Co

gel: lane Co, 500 jcg pBR322:

lane a, Co + 100 pmol Cr(lIl):

lane b. Co + 100 pmol Cr(VI); lane c. Co + 100 pmol Cr(V1) + 200 nmol GSll: pmol Cr(V1) + 9.8 /tmol H302; lane cl. Co + Y.8 /tmol H,Oz.

h

j Fig. 2. Ethidium-bromide + 980 nmol H& Co +

stained

g

agarose

f

+ 9.8 jmol

Co

gel: lane Co, 500 ,ug pBR322;

lane f. Co + 100 pmol Cr(VI)

100 pmol Cr(V1)

d2

lane d2. Co + 100 pmol Cr(V1)

+ 9.8 ,umol H1O? + supcroxide

I1@2 ,n DzO; lane h, Co + 100 pmol Cr(IIl)

lane j. Co + 200 nmol GSH

lane dl. (‘o + 100

+ 9.8 /on01 HLOI.

dismutase;

lane g.

+ 9.8 ,umol H?O,:

299

Start

co2 Fig. 3. Nick translation

d2C

assay (autoradiogram):

b

a

lane Cal.

co1 500 pg pBR322

+ DNA polymerase;

Co + 100 pmol Cr(II1); lane b, Co + 100 pmol Cr(V1); lane c, Co + 100 pmol Cr(V1) GSH; lane d2. Co + 100 pmol Cr(V1) + 9.8 nmol HzOz; lane Co2, Co + DNase

+

lane a, 100 nmol

I + DNA-polymerase

I.

protocols dl, d2 and g (see Materials and Methods), revealed the nicked circular form (cc-form) of the plasmid. This finding indicates that DNA strand breaks were formed after reduction of Cr(VI) by H202 in Hz0 was well as in D20. Furthermore, a new band, the linear form, could be seen. This minor band was slightly intensified, however, after incubation without Cr(V1) but with GSH and H?O? (lane j, Fig. 2). Lane f indicates that the addition of superoxide dismutase prevented the occurrence of DNA strand breaks. Similar resuits were obtained using the radioactive nick translation assay as an indicator for DNA strand breaks. A typical autoradiogram obtained by this assay is shown in Fig. 3. Two control incubations were performed in each experiment (lanes Co1 and Co2). Lane Co1 shows the incorporation of radioactively labelled dNTP into the plasmid after incubation with DNA-polymerase I (background). This incorporation of radioactivity was a result of the presence of the nicked form which originated during preparation of the plasmid. Lane Co2 shows the incorporation of radioactivity into the plasmid DNA after incubation with DNase I and DNA-polymerase I, the typical nick translation reaction. No alteration of the assay reaction could be detected after adding Cr(III) or Cr(VI) (lanes a and b). In addition, reduction of Cr(VI) by glutathione did not induce DNA strand breaks exceeding the background effect (lane c). However, when Cr(VI) was reduced by H102, higher amounts of radioactive dNTP were incorporated into plasmid DNA by DNA-polymerase I, indicating that the presence of DNA strand breaks after this reduction process (lane d2, Fig. 3) represents incubations according to the same indexed protocols (see Materials and Methods).

Using the nick translation assay with radioactive deoxyribunocleotide triphosphate and agarose gel electrophoresis, the reactivity of two different chromium species with the double-stranded plasmid pBR322 was studied. The results clearly indicate that Cr(VI) was able to induce DNA strand breaks only when reduced by H:Oz. D# did not influence the occurrence of a responsible reactive intermediate (lane g, Fig. 2), but the occurrence of the reactive intermediate was abolished by the addition of superoxide dismutase (lane f, Fig. 2). No DNA strand breaks could be detected when Cr(VI) was reduced by GSH in excess (lane c, Fig. 1). DNA damage is a critical event in the initiation of carcinogenesis. In the case ol carcinogenic chromates. DNA single strand breaks [I 31 and cross-linking of nuclear proteins to DNA [ 121 have been reported. It is of great interest to elucidate the nature of the reactive chromium species responsible for chromate-induced genotoxicity and carcinogenesis. It has been previously shown that Cr(V) is produced during reduction of Cr(VI) by microsomcs [ 171. GSH [I81 or ascorbatc [l9]. It has further been demonstrated that highly reactive hydroxyl radicals are generated by reduction of chromates through H?O: [20]. It rctnains to be clarified which of these reactive compounds causes the DNA strand breaks that are observed after exposure of cells to chromate [ 131. The results of the present study do not favour Cr(V) as a DNA strand-breaking agent. This is demonstrated by lane c in Fig. I. Using superoxide dismutase. singlet oxygen could be excludcd from being responsible for the DNA damage indicated. Cl-(V). which can bc monitored by ESR. can be detected as a metabolite of Cr(VI) only in the presence of a slight excess of reducing agents [ 181. However. under physiological conditions. transitory Cr(V) can be expected to react immediately with GSH (which is intraccllularly present at concentrations of about 5 7 mM). IJnder such conditions. Cr(V) is present for only an extremely brief interval [2l]. On the other hand, it has also been proven that hydroxy! radicals which are generated by ionizing radiation can cause DNA damage [22. 231. In peroxisomes. fatty acids are mctabolircd by stepwise rcmoval of two-carbon units. EGch step generates one molecule of Hz02 which possibly may cause reduction of Cr(V1) [24, 351 by a mechanism which involves superoxide anions and.hydroxyl radicals. The results of this investigation are therefore consistent with the assumption that superoxide anions and hydroxyl radicals resulting from decay of peroxochromium(V) complexes, rather than Cr(V) itself, are the reactive species responsible for specific DNA damage observed during reduction of Cr(VI). REFERENCES

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