Induction of sfiA SOS function by peroxides using three different E. coli strains

325

TXL 02204

Induction of sfiA SOS function three different E. coli strains

Erwin Eder’, Alain Favre2, Claudia Christoph Deininger’

(Received

I I January

(Revision

received 21 April 1989)

(Accepted

24 April 1989)

by peroxides using

Stichtmann’

and

1989)

Kc?, b~,ort/,s:SOS-.sfrA-induction:

Peroxides;

E. co/i strains

SUMMARY Five peroxides 3 different

and two related

E. co/i strains

tylhydroperoxidc. obtained function

cumene hydroperoxide)

from the different

of excision

repair

compounds

were tested for genotoxocity

(PQ37. PM21, GC4798). strains

nor covalent

by hydroperoxides.

binding

of radicals

that neither compounds

Paraquat

activity.

When using strains

was inactive

PM21 and GC4798.

peroxide,

tested,

using /rr/-bu-

of results

From a comparison

DNA lesions lading

to DNA is responsible

the remaining

clearly positive result in strain PQ37 whereas di-tert-butylperoxidc borderline

(hydrogen

were clearly positive in all strams.

it can be concluded

Among

by the SOS Chromotest

All tested hydroperoxides

to the induction

for the induction

of .s/rA-SOS

only dibenzoylpcroxide

and azobisisobutyronitrile none of the latter compounds

gave a

showed only was positive.

in all strams.

INTRODUCTION

Hydrogen peroxide, organic peroxides and hydroperoxides are frequently used industrial chemicals, e.g. as a source of free radicals in the plastic and rubber industry, as bleaching agents for foods and paper pulps, in the production of textiles. cosmetics and pharmaceuticals, and as chemical intermediates [ 11. Possible mechanisms such as interaction with DNA which could lead to genotoxicity, mutagenicity and carcinogenecity by this class of compounds have been proposed Address

for correspondence:

Strasse 9, D-8700 Wiirzburg.

037%4274:X9/33.50

Erwin Eder. Institute

of Toxicology.

University

of Wiirzburg,

F.R.G.

(” 1989 Elscvier Science Publishers

B.V. (Biomedical

Division)

Versbacher-

((21 and references

therein).

Nevertheless,

the data available

from the literature

on

the genotoxic. mutagenic and carcinogenic effects are still insufficient and require further investigation before a general understanding of these biological effects can be obtained [36]. Peroxides were not mutagenic in the Ames test when using standard test strains TA1535, TAIOO, TAl53X or TA98 [3, 4. 61. However. with the newer strains TA I02 and TA2638 which possess the hi.sG428 mutation. a slight mutagenic response could be observed with some hydroperoxides [3]. We have now investigated the SOS-inducing activity of several peroxides and related compounds with 3 ditfcrent E. co/i strains (PQ37, PM21, GC4798) which contain the .s/iA::/uc,Z gene fusion [7]. Strain PQ37 is a frequently used standard test strain [Xl. Strain PM21 is derived from ABI I57 and contains the IIUVA mutation responsible for a deficiency in 4-thiouridinc, which normally behaves as an antiphotomutagenic agent [9, IO]. The strain was recently successfully used to detect genotoxic effects of dioxetanes [I I] and monofunctional methanesulphonates [l2]. Strain GC4798 [I31 is also derived from ABI I57 and shows high sensitivity to alkylating compounds [l4]. in particular to those compounds tending to N-alkylation in the nucleobases [I21 due to their lack of the repair enzymes, ;V-3-methyladenine-DNA glycosylases. A comparison of the results obtained with these different strains may elucidate the underlying mechanisms involved in the observed genotoxic effects. MATtRIALS

AND METHODS

The chemicals and reagents used were purchased from Merck. Darmstadt (F.R.G.), Aldrich, Steinheim (F.R.G.) or from Sigma, Deisenhofen (F.R.G.). They’ were of the highest purity available.

The sources. isobutyronitrile

structures and purities of the substances and paraquat are listed in Table I.

tested.

5 peroxides,

azobis-

Due to differing solubilities of the test compounds, 3 different solvents had to be used for dilutions of the test substances. Hydrogen peroxide, tllrr-butylhydroperoxide and paraquat were dissolved in water, cumene hydroperoxide. dibenzoyl peroxide and azobisisobutyronitrile in DMSO and di-trrt-butylperoxide in ethanol. BrrctcJrial .struitl.s Strain PQ37 has been described by Quillardet and Hofnung [S]. Strain PM21 is derived from E. co/i K 12 AB I 157 (t/w I ICU-6 proA his-4 argE3 thi- I IucY 1 ~ulK2

221

TABLE

I

STRUCTURE.

SOURCE

AND PURITY

Substance Hydrogen

peroxide

OF THE TESTED

COMPOUNDS

Structure

Source

HOOH

Aldrich

Purity 30% residue Hz0 (titration,

/PY~ Butylhydroperoxide

Aldrich

+OOH

KMnO,)

70% residue Hz0 (iodometric ‘H-NMR

Di-tcrr

butylperoxide

Aldrich

+oo+

titration, spectroscopy)

98% (gas chromatography)

Cumene DibenLoyl

hydroperoxide

’ c

+ OOH

Sigma

80% (iodometric

titration)

peroxide Aldrich

97%

Aldrich

98% (according

(iodometric Azobisisobutyronitrile

CN+NN+CN

titration) to indication

of supplier) Paraquat

CH,-QN3@N”

CH,

Aldrich

99%

(I, 1-dimethyl-4,4-

(potentiometry

bipyridinium

AgNOI)

dichloride)

with

~~a-14 .YJ+~ mtl-I tsx-33 strA3 I supE44) by introduction of the nuvA mutation (4-thiouridine thiolase deficiency) and introduction of sfiA::lacZ gene fusion [9]. Strain GC4798 was constructed by Boiteux et al. [13]. It carries the same markers as ABI 1.57 (see above) and additionally X:: Tn 5 tugA alkAl (p($A krc)CIind). The tugA gene codes for 3-methyladenine-DNA glycosylase Tug1 and ulkA for 3-methyladenine-DNA glycosylase TagII. Determinution

of’SOSIP

values when using strain PQ37

The test procedure (including media and overnight culture) was performed as described by Quillardet and Hofnung [8]. The SOSIP values were determined from the linear part of the slope of the SOS induction factor (I) dose-response curve according to the method of Quillardet and Hofnung [S]. The induction factor I(c) is the ratio I(c) = U,,,/U,) and R(c) =fi-gal units/alkaline P-ase units. R(o) is the background value using only the solvent. Mean values were calculated from 3 independent determinations. The differences were less than 15%. Determination qf Ijnmol (SOSIPJ bvhen using strains PM21 und GC4798 Because strains PM21 and GC4798 do not contain the PhoC marker utilized in strain PQ37 for the constitutive expression of alkaline phosphatase, the toxicity correction of these strains had to be performed via the overall toxicity determined from cell growth delay as measured by absorbance at 600 nm (OD6&.

228

~l/lcciirr rir~tl.sol~rtioii.r

,MciZ /llc,t/ilrtll sol\ed

13.6 g KHzP03,

in 1 liter water and adjusted

and 2 g gl~~cose IO mg thiamine

the solution were added.

2 g (NH&SOJ and 0.5 mg FcS03.7H70 were diswith KOH to pH 7.0. After addition of4 g casein

was autoclavrd

for 20 min. Then 200 mg MgSOJ and

Bz@r H 0.75 g KC’I. 17.5 g Na2HPOJ.2H,0. 5.5 g NaHzPOI.H20. 0.25 g MgSOl 7HzO. 3.7 ml jj-mercaptoethanol and I g SDS were dissolved in I liter water and adjusted to pH 7.0 with KOH. ONPG’ .so/zrtior~ 400 mg ONPG acre dissolved in 100 ml phosphate buffer. pII 7.0, and stirred in the absence of light at 4’ c’. ;V(rJCO., .solntiotr I Oh g Na,C‘O? were dissolved in I liter water.

50 /rl of stock solution of the bacterial culture at 17 (’ in an incubator shaker for I? h.

were incubated

in 5 ml M61 medium

200 /II of the overnight c culture were diluted with IO ml M63 medium and the mixturc was again incubated at 37 (’ until an OD h,Io of about 0.6 was obtained. 5 m1 of this culture Lver-ediluted with 20 ml M63 medium and 1.2 ml aliquots of this SLISpension were pipetted into sterile test tubes. After addition of the test substance in 40 111solutions of the appropriate solvent. the mixture was further incubated at 37 (’ for about 2 h. As control 40 ,~tl of the pure solvent were added instead of test substance solutions in order to determine the background. The ODhoo of each sample was measured immediately aftcr the end of the incubation.

100 ,d of the bacterial

suspension (after incubation with the test substances, see above) were rigorously mixed with 0.9 ml buffer B containing 3 drops ofchloroform for complete lysis of the bacteria in order to release the /I-galactosidase and 200 ill of the ONPG solution were incubated for IO 90 min at 37’C (the 0D411) should be about 0.5). The /I-galactosidase is then inhibited by addition of666 ,LLINa2C03 solution and the absorbance of the mixture is measured at 420 nm (OD420). In addition. each sample is tneasured at 550 nm (OD550) in order to correct for light scattering due to cell fragments (see formula of Miller below). The relative /I-galactosidase activity (U) is calculated according to the formula of Miller [ 151:

C’uIc~~l~~tiot1 of’/~t~~~oli SOSIP)

The Enmol

(= SOSIP)

wlucs ~t.Jwi~ using .stsuin.s PM21 untl ti(‘479N

values were calculated

in accordance

with the method

ol

229

Quillardet

[S]. For better differentiation,

for the results obtained

in Table

with PQ37 (Hofnung)

with PM21 and GC4789. The ‘induction factors’

II the term ‘SOSIP value’ is used

and ‘I/nmol’

for a given concentration

the relative /&galactosidase

activity

for the results

I(c) were calculated

U(c) at a given concentration

obtained

by dividing

(c) with the back-

ground P-galactosidase activity (U,,). I(c)=U~,,/U,. The I(c) values were plotted against the dose and the linear slope of the doseeresponse curve was used for calculation of the SOSIP = 1,‘nmol of the test substance. Again the mean values of at least 3 independent determinations were calculated. In all cases the differences were less than 15%. RESULTS

The SOS inducing potencies. the maximal induction factors and the highest P-galactosidase activities (P-gal for PQ37 or U for PM21 and GC4798, respectively) are summarized in Table II. All hydroperoxides were clearly positive in all 3 strains. According to Quillardet and Hofnung [8], compounds are considered as significantly genotoxic if I,,,;,, is at least 1.5. When strains PM21 and GC4798 are utilized, a substance is considered genotoxic if the relative p-gal U is at least 1.5 times the background (normally 60 ~70 for both strains) irrespective of the dose. The SOSIP values of the hydroperoxides are not so high as that of nitroquinoline oxide (NQO) for which we measured a SOSIP of 26. They are, however. in the range of values ob-

TABLE

II

SOS INDUCING MAXIMAL RELATLD

POTENCY

Substance

Hydrogen

MAXIMAL (b-gal)

PO37

peroxide

tc’~f-But~lhSdroperoxide Ctnnenc

(SOSIP=I’nmol).

INDUCED /I-GALACTOSIDASE COMPOUNDS

hydroperoxide

INDUCTION

ACTIVITIES

PM21

GC479X

7.X

0.026

X.6

796

0.01 3

X.6

52i

6.5

X.6

0.053

7.X

412

0.03

7.x

515

5.1

5.1

0.07

s.0

133

0.072

5.X

404

I .49

1.22

0

1.1

42

0

1.1

55

2.3

s.0

0

1.1

X3

0

I.1

74

I .59

2.02

0

1.2

72

0

I.1

X0

1.2

4.2

0

1

42

0

I

48

0.04

Dlbenzoylperoxidc

0.02

Arobl\isobutyronitrilc

5.6x0

Paraquat (meth\Iviologenc)

0

’

IO 4

~______

h/l-Galactosidasc

according

~Enrqme units according

AND

0.01

4.3x

,‘Multiple of the background

(I,,,,,,) AND

0.022

Di-fc,,-t-butylpcroxide

35.x

FACTORS

(U,,,,,,) OF PEROXIDES

.______ I(o)=

I (see Methods).

to Quillardet to the formula

and Hofnung

[Xl.

of Miller [I51 (background

60 X0).

__

230

tained

with strongly

alkylating

compounds

such as methylmethane

isopropylmethane sulphonate [ 161. Cumcne hydroperoxide molecular weight (and molecular si/c) of the hydroperoxidcs est SOSIP in all strains whereas hydrogen displayed the lowest activity in all strains.

sulphonate

ot

possessing the highest tested yielded the high-

peroxide with the lowest molecular Cc No significant differences in SOSIP were

observed in the difrercnt strains for the hydroperoxides tested, indicating that the characteristic design of the strains. e.g. the lack of excision repair (PQ37) or the lack of :V-3-methyladenine-DNA glycosylase. does not signiticantly influence the induction of SOS repair. Among the dialkyl peroxides. diarylperoxides and r&ted conpounds. onI\, dibenLoylperoxidc showed a clear SOS inducing activity (SOSIP) in strain PQ37 according to the definition of Quillardet (set above) but not in the other There strains (PM2 I and GC4798). The I,,,,, is 2.3 times higher than the background. is. however, only a slight dose-dependent increase in /Cgalactosidasc activity and the rise in the induction factors in due mainly to the toxicity correction via alkaline pho\phatasc which significantly decreases even at rather loti doses (see Fig. 1). Di-/cr/butylpcroxide and ~lzobisisobutyronitrilc, which was inclued in this series due to it\ structural similarity to di-/err-butylperoxidc and bcca~~se it is a frequently used radcal-producing agent, both showed only borderline activity. They possess maximal induction f‘actors of 1.59 for azobisisobutyronitrile and I .40 for di-rc~l/-butylhydrc,peroxide and arc to be considcrcd as genotoxic according to Quillardct‘s criteria. It must bc mentioned. howevcr. that the SOSIP values of 4.3 x IO ’ for di-tc)f/-butylperoxide and 5.6 x IO ’ for a/obisisobutyronitrile were cxtrcmely low. Paraquat \vas

231

included

in this work because

it can readily

produce

genotoxic

radicals

superoxide

be reduced

in the presence

to stable radicals

which may

of oxygen [ 171. No SOS induc-

ing activity could be observed for paraquat in the PQ37 strain. Absolutely no SOS inducing activity could be found for di-[err-butylperoxide, dibenzoylperoxide, azobisisobutyronitrile or paraquat when using strain PM21 or GC4798. DISCUSSION

One of the most interesting results obtained in this study is that all tested hydroperoxides possess clear SOS inducing activity in all test strains whereas the alkylperoxides and related compounds are much less active or inactive. It is well known that the hydroperoxides produce hydroxyl radicals by spontaneous cleavage or by interaction with metal complexes. e.g. Fei+ m the Haber-Weiss process [2]. Hydroxyl radicals show a high tendency to oxidize thymidine leading to oxidized products such as thymidine glycol, 5-hydroxymethoxyuridine and they are known to induce strand breaks [ 18. 191.The proposed interaction of hydroperoxides with thymidine is in accordance with the fact that hydroperoxides show mutagenic activities in strain TA102 [3] which carries the lzisG428 nonsense mutation TAA/ATT and the alteration of thymidine can lcad directly to back mutation [20]. When using strain TAlOO (in which a GC-AT transition leads to back mutation), the hydroperoxides did not show mutagenic activity. At first glance it is surprising that the hydroperoxides do not induce a mutagenic effect via the pkM101 plasmid in TAIOO although a clear SOS induction was found in the E. co/i tests strains. Probably the DNA lesions caused by hydroperoxides. e.g. thymidine oxidation. can induce the ?fiA function in the E. cdi test strains. but they clearly do not contribute significantly to the induction of errorprone repair via the pkMlOl plasmid in S. typhimuriurn TAlOO as was clearly found for the strongly alkylating methylmethane sulphonate [12, 21, 221. Recently Cerutti [I 71 very briefly speculated that ‘it is conceivable that prooxidant states induce a group of prooxidant genes reminiscent of SOS functions in bacteria’. He did not, however. make further comment on this sentence and did not state which of the genes in the cascade leading to SOS repair he meant. From our results no indication can be derived that such processes are relevant to the induction of .$A function. A further interesting result is that there are no clear differences in the SOS inducing potencies of the tested hydroperoxides in the 3 strains, although the strains differ in characteristic genetic markers as regards susceptibility to specific DNA lesions. When considering our results from this point of view, some interesting conclusions can be drawn: (1) Evidently the tested organic hydroperoxides can easily pass through the intact bacterial envelope since there is no difference in genotoxicity between bacteria PM2 I and GC4798 which do not possess the yfumutation andPQ37 which carries the $u mutation.

232

(2)

No

seems

DNA

lesion

which

to be involved

difference r/r~A

is found

mutation repair

in the gcnotoxic

in genotoxicity

and those

as the UV-induced cision

cyclobutanc

(3) A covalent

binding

in GC47OX

alkylating glycosylssea peroxides

latter

Dependence The

and cumenc

of SOS

higher

inducing

doa

rcsponsc

(i<‘479X

ol‘cx-

lesion\. not occur OI-

since genc)to\-

is highly

hcnsiti\c

by the fact that

strains

t/71,p/7ir777rri7rn7

activity

Thus

substances

on molecular

TA

to

the hydra)-

1535 and TA

would

components.

Alkoxy

radicals

greater

lesion.

Evidence,

100 w hich

degree of spontaneous

molecular greater

however.

in all .;

of hydroxyl

homol\ tic

radicals

could

weight. binding

molecular

was found

does not occur or does not contribute

was observed

of /c,t-butylhydrop~r(~~i~i~

concentration

bc the covalent with

weight

substitution

a higher

of higher

explanation

binding

such

due to its lack of ;v:-adentne-DNA

may lead to ;I higher

elTccts.

Another DNA

DNA

such

the

distortion

compounds.

hydropcroxide with

helix

components

strains.

containing

for the induction

of the SOS

is conlirmed

degree of unsytnmetric

cleavage due to steric be produced

in S.

to alkylating

a highcr

system

since no clear

(PQ37)

do not form

binding)

conclusion

strain

is required

to DNA

than in the other

are not mutagenic

are also scnsitivc

strains.

The

dimer

radicals

DNA

repair

of these hydroperoxidcs

to the induction

(covalent

I?].

by the excision

it. In general

thymidine

of alkoxy

is no higher

[I?

action

the hydropcroxidcs

contribute

compounds

repaired

bctwecn the bacterial

not possessing

[23]. Presumably

does not significantly icity

can be readily

of alkoxy size

in our

radical\

would study

to the induction

then

to IIN: produce

that alkoxy of the SOS

;I

radical rcsponsc

(see above). The

other

species,

tested peroxides

e.g. by transfer

molecule,

tcrial

Three

envelope

a

dccisicc

PQ37

(OD,,,,~)

more

role (strain

leads

to ditTcrcnt

carries

on the basis

of our

results

(a) and (b). explanation

(c) deserved

vised

positive

in cases in which

pcroxidc

in this

PQ37

contains

mechanism

the u~rA

case

to the oxygen

but not in the other in genotou-

through

the

the ~fir mutation. in which

mutation)

excision

is operating

via alkaline

hat-

than repair

in the C;ISC

phosphatase

by correction

C‘OE

onlb one con-

the difference

dibenzoylpcroxide

which

oxygen

can he further

[2]. Surprisingly

in strain

correction

results

intermediates

can be given for

genotoxic

rcactivc

as u\cci

via the cell densit!

(see Methods).

Whereas

increase

radical

dismutase

in PQ37.

(c) the toxicity

may also form

by these processes

of the bulky

readily PQ37

their

genotoxic

explanations

(b) an additional

of diben7oylperoxide: with

was slightly

(a) penetration

occurs

strains:

from formed

by superoxide

possible

in these strains:

in the other plays

electron radical

peroxide

dibenioylperoxide.

two strains. icity

of an

and the superoxidc

verted to hydrogen pound.

and related compounds

in /I-galactosidase a slight

activity

but reproducible

nothing further

SOSIP

can be said to date about

consideration.

values

are obtained

can be measured dose-dependent

In general,

possibilities

caution

is ad-

but no dose-dependent

[ 12. 241. In the case of diben/o\;lincrease

in /I-galactosidase

actor It!

233

(Fig.

1) was found:

in alkaline

the high SOSIP

phosphatase

activity

value, however,

resulted

from the steep decline

{Fig. I). Due to the reproducible

increase

in /3-galac-

tosidase activity dibenzoxylperoxide should be considered as unambiguously genotoxic in SOS Chromotest strain PQ37. The high SOSIP value, however, should be cautiously interpreted. As we have demonstrated in previous studies [ 12, 161, the different toxicity correction could lead to misinterpretations in borderline cases in which no or only a marginal increase in /I-galactosidase activity is observed (a more detailed discussion of these phenomena was given recently by Eder et al. [12]). To our knowledge no mutageilic activity of dibenzoyiperoxide has been reported in the literature; it is considered, however, to be a tumour-promoting agent [5]. No clear-cut explanation can be given to date as to why di-tert-butylperoxide, ~1zobisisobLltyronitrile and paraquat do not show a clear SOS inducing activity. Only in strain PQ37 did we find a borderline response (see Results). In general. the situation is similar to that in mutagenicity testing. Mutagenic activities have been obtained for hydroperoxides when using strain TAlO2 or TA2638, but no activity or negative results have been reported for dialkyl(aryl)peroxides, azobisisobutyronitriie and paraquat [3]. The only report that paraquat is mutagenic in the Ames test using strain TAIOO [25] has been contradicted [26]. Ruiz-Rubio et al. [26] found, however. a positive result for paraquat when applying a forward mutation test system (L-arabinose resistance) using S. r~/~~?~~lzi~jlf~?z strain BA9. ACKNOWLEDGEMENTS

We are indebted to S. Boiteux for his kind gift of strain GC4798. We wish to thank Mrs. C. Grimm, Mrs. E. Weinfurtner and Mrs. D. Muth for excellent technical assistance. This work was supported through ‘Procope’ in a joint research project between France and the Federal Republic of Germany, the ‘Deutsche Forschungsgemeinschaft’ (SFB 172) and the ‘Bundesministerium fiir Forschung und Technologie’. REFERENCES

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