relationship between dna damage and survival in formaldehyde-treated mouse cells

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Mutation Research, 79 (1980) 277--283 © Elsevier/North Holland Biomedical Press

RELATIONSHIP BETWEEN DNA DAMAGE AND SURVIVAL IN FORMALDEHYDE-TREATED MOUSE CELLS

W.E. ROSS and N. SHIPLEY

Division of Medical Oncology and Department of Pharmacology, University of Florida College of Medicine, Gainesville, FL 32610 (U.S.A.)

(Received 8 February 1980) (Revision received 2 May 1980) (Accepted 9 June 1980)

Summary

Mouse cells exposed to formaldehyde (FA) were examined for colony- forming ability and DNA synthesis as well as the induction of DNA damage. Cell survival was unaffected until the FA concentration exceeded 150 #M after which there was an exponential decay in survival. Formaldehyde-treated cells studied by the DNA-alkaline-elution technique exhibited a low frequency of single,strand breaks but extensive DNA--protein crosslinking. These cross- links were repaired following removal of the drug. The DNA damage was accompanied by inhibition of DNA synthesis.

Formaldehyde (FA) is ubiquitous in man's invironment. It is widely used in industry and agriculture and is a product of combustion of automobiles and cigarettes [1]. Genetic efffects of FA were initially described in 1946 [14] and since that time its mutagenic activity has been demonstrated in bacteria [12], Neurospora [4], yeast [2] and Drosophila [1]. While the risk of FA at low exposure levels to human is unknown, its mutagenicity in other systems has generated renewed interest in the effects of FA on DNA. DNA-strand breaks and DNA--protein crosslinks have been described in E. coli [12] and Sac- charomyces cerevisiae [10,9]. Single-strand breaks have been ascribed to DNA- excision repair [10] and are not considered a direct effect of the FA. On the other hand, the DNA--protein crosslinks are almost certainly a direct effect of FA, but their relationship to cytotoxicity and mutagenesis is unknown. To date, there is little information regarding the effects of FA on the DNA of mammalian cells [11]. For this reason we have examined mouse L1210 cells

Suppor ted b y NCI Research Grant C A - 2 4 5 8 6 and RCDA CA-00537 (W.R.).

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after treatment with FA. Using the DNA-alkaline-elution technique, we have been able to study FA-induced strand breaks and DNA--protein crosslinks and their relationship to DNA synthesis and cytotoxicity.

Methods

Mouse leukemia L1210 cells, grown in suspension in RPMI 1630 medium with 20% fetal calf serum, were employed in all experiments, Details of tissue culture technique and the labeling of cells with radioactive thymidine have been published [7]. Colony-forming ability was determined using the soft-agar colony-forming technique of Chu and Fisher [3].

Formaldehyde solution * (Fisher, 37%) was diluted in sterile H20 prior to use. 1 h prior to adding FA, cells were resuspended in fresh warm medium at 5 × l0 s cells/ml. Drug treatment was for 2.5 h after which the cells were washed twice with fresh cold medium-and resuspended. When cells were studied for repair of DNA damage, they were allowed to incubate in fresh medium at 37°C for various intervals after drug removal.

The theoretical basis for and technical details of DNA alkaline elution have been described elsewhere [7]. Briefly, L1210 cells containing 14C-labeled DNA are treated with FA, washed with cold medium and layered onto a polyvinyl chloride filter along with untreated cells containing 3H-labeled DNA which have been irradiated with 150 R. These 3H-labeled cells serve as an internal standard. In order to measure single-strand breaks, cells are lysed in the presence of pro- teinase K and the elution carried out as previously described [7]. The rate of elution of the DNA from the filter is directly related to the frequency of strand breaks. For measuring DNA--protein crosslinks a similar protocol is followed except that, following FA treatment, cells are given 300 R or X-ray and cell lysis is performed without proteinase K. Because protein adsorbs to the filter under standard eluting conditions the presence of DNA--protein cross- links retards the rapid elution of irradiated DNA from the filter [8]. The degree to which the DNA is retarded is dependent on the frequency of the DNA--pro- tein crosslinks. DNA interstrand crosslinks, by increasing the apparent strand length, also reduce the effects of X-ray on DNA elution [5]. This effect is not reversed by proteinase K thereby providing a means of studying the 2 indepen- dently.

Synthesis of DNA was measured by incorporation of [3H]thymidine into TCA-precipitable DNA. After removal of FA, cells were incubated for 30 min in the presence of [3H]thymidine. Incorporation was stopped by precipitating the cells with 5% TCA and collecting the precipitate on glass filters. Radioactiv- ity was solubilized from the filter by adding 0.4 ml of HC1, 1 N, and incubating at 60°C for 1 h after which 2.5 ml 0.4 N NaOH was added. After standing in room temperature for 1 h, liquid-scintillation fluid was added and the counts obtained.

* Th i s s o l u t i o n c o n t a i n s 12 - -15% m e t h a n o l . S tud ied i n d e p e n d e n t l y , c o m p a r a b l e c o n c e n t r a t i o n s of m e t h a n o l p r o d u c e d n o D N A d a m a g e .

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Results

Formaldehyde cytotoxicity was studied by exposing L1210 cells to various FA concentrations for 2.5 h, removing the drug, and then seeding the cells in soft agar (Fig. 1). The survival curve demonstrates a large shoulder, after which there is a precipitous decrease in colony-forming ability over a concentration range of 175--250 #M. In other experiments we have foumd that elimination of serum during FA treatment reduces the shoulder on the survival curve (data not shown).

Production of DNA single~trand breaks following FA treatment was assayed by the alkaline~elution technique. After FA treatment, cells were lysed on the filter in the presence of proteinase K to remove any DNA--protein crosslinks which might obscure the presence of strand breaks (Fig. 2B). A small number of strand breaks are apparent at 200 pM and these increase as the concentration of FA is increased to 300 pM.

The alkaline-elution technique provides a simple means of documenting the presence of DNA--protein crosslinks following treatment with FA. Fig. 2A shows the effects of various concentrations of FA on elution of irradiated DNA

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280

STANDARD LYSIS LYSIS WITH PROTEINASE K

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to FA. Fo l lowing r e m o v a l of FA, cells we re lysed on t he e lu t ion fi l ter in the absence (A) or p resence (B) of p ro t e inase K and e lu ted f r o m the f i l ter acco rd ing to Methods . E lu t ion of e x p e ~ n e n t a l [ 1 4 C ] D N A is p l o t t e d against the s i m u l t a n e o u s e lu t ion of in t e rna l s t a n d a r d [ 3 H ] D N A . S o m e e x p e r i m e n t a l cells r ece ived 300 R X- i r rad ia t ion on ice p r io r to e in t ion .

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281

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from the filter. Over a concentration range of 10--100 #M, there is a progressive decrease in the rate of elution of DNA from the filter suggesting the presence of crosslinks. If the same cells are lysed in the presence of proteinase K (Fig. 2B), the crosslinking effect is completely reversed confirming that the cross- links were of the DNA--protein type.

Since DNA--protein crosslinks occur at relatively non-toxic doses (i.e., less than 200/~M), it was of interest to determine if these crosslinks were repaired. Following treatment with 100 /~M FA, cells were incubated in fresh warm growth media. At various times, aliquots were removed and assayed for the presence of DNA--protein crosslinks (Fig. 3). There'was a progressive loss in crosslinking effect after FA removal suggesting that these lesions were repaired. The repair process appears to be complete 24 h after drug removal.

The effect of the described DNA damage" on thymidine incorporation into DNA is shown in Fig. 4. As the concentration of FA is increased from 50 to 200/~M, there is a progressive decline in thymidine uncorporation.

Discussion

Our data in L1210 cells demonstrate that at relatively non-toxic FA concen- trations extensive DNA--protein crosslinking occurs which is repaired over a period of time following of the drug. DNA-strand breaks are also observed but only after treatment with more toxic FA concentrations. These data are in general agreement with those described in E. coil [13] and yeast [9,10].

Colony-forming ability following FA treatment exhibits a threshold effect such that there is little inhibition of colony formation at doses less than 150 #M. There is a clear discrepancy in the dose--response relationship when survival is considered as opposed to DNA--protein crosslinking or DNA- synthesis inhibition for which there is little evidence to suggest a threshold. This discrepancy has been previously observed in yeast [9]. There are at least 2 theoretical explanations to account for this phenomenon. First, the DNA-- protein crosslinks are non-lethal at lower FA concentrations because of DNA

282

repair and that only after the repair processes are saturated is the damage expressed as lethality. Martin et al. [11] have demonstrated unscheduled DNA synthesis in HeLa cells following t reatment with FA and in this work we provide direct evidence for the first t ime that FA-induced DNA--protein cross- links are removed. Further work will be required to clarify the relationship between this repair and the ability of the cells to survive the crosslink. In addi- tion, it is important to note that the alkaline-elution technique, while a superior tool for detecting DNA--protein crosslinks, does no t allow discrimina- tion between protein binding to a single DNA-strand or protein-mediated DNA- interstrand bridging. Such qualitatively different forms of DNA--protein cross- linking may have vastly different consequences with regard to cell survival and may no t occur with a simple linear relationship to FA concentration. A second explanation for the discordancy between observed DNA damage and survival may be that other forms of DNA damage are responsible for lethality. Intra- strand and intrastrand crosslinking of DNA can theoretically occur following FA exposure [6]. Intrastrand crosslinks are not detectable by the alkaline-elu- tion method. Wilkins and Macleod [16] provided evidence of interstrand cross- linking in E. coli following FA treatment but we have been unable to document such lesions in our experiments.

DNA-strand breaks following exposure to FA have been at t r ibuted to DNA- excision repair and, indeed, appear to occur less frequently in repair~leficient mutants of S. cerevisiae [9]. Our data in L1210 cells are consistent with this hypothesis. These mouse cells are deficient in excision repair and we find that strand breaks are no t extensive and occur only at toxic FA concentrations. Our data do not allow us to state with certainty whether the mechanism of break formation is enzymatic or direct damage by FA.

Finally, our data suggest that FA may provide a useful tool for examining the significance of DNA--protein crosslinks in man. These types of lesions are also caused by a variety of drugs used in cancer therapy [5,15,17] but their significance with regard to therapeutic efficacy has been obscure to date.

Acknowledgement

The authors wish to thank Ms. Linda Bell for her secretarial assistance in the preparation of this manuscript.

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