Chtm.4iol. fnleruclions, 13 (1978) 173-179 vier Scientit& Publishi Company, Amsterdam - Printed in The Netherlands

CYTOTOXICITY, MUTATIONS AND DNA DAMAGE PRODUCED IN STER CELLS TREATED WITH STREPTOZOTOCIN, ITS

ANALUGS, AND Af-METHYL-A/‘-NITRO-iv-NITROSOGUANIDINE *

B.K. BHUYAN ‘. AR. PBTBRSGN b and CHARLES HEIDELBERGER b

&. The Upjohn Company, Kalamazoo. Mich. 49001 and b McArdle Labsralory for Cancer Rewwch. University 01 Wisconsin, Madison, Wis. 53706 (U.S.A.)

(Received dune 3rd. 1975) (Revtaion received August 30th, 1975) (Accepted October 23rd, 1975)

The activities of streptozotocin (SZ), three structural analogs of SZ, and N-methyl-N’nitro-N-nitrosoguanidine (MNNG) in producing cytotoxicity, mutations to 8-azaguanine (8-AzG) resistance, and DNA damage (single- strand breaks) in V79 Chinese hamster cells have been examined. These three biological processes appear to be associated. MNNG was about lo3 times more active on a molar basis than SZ, md the activities of the analogs fell within these extremes.

INTRODUCTION

SZ is an antibiotic with broad spectrum antibacterial [l] , and with anti- tumor 12.31 and diabetogenic activity [3]. It is useful in the treatment of patients with malignant islet-cell tumors [4]. The structure of SZ contains a methyl nitroso group, and in this respect it is related to the methylating agents, NMU and MNNG, which are powerful mutagens and carcinogens [5].

* This work was supported in part by grant CA-07175 from the National Cancer Insti- tute, National Institutes of Health. Abbreviations: 8-A&l, 8ataguanine; [ 14C]TdR, [ 2.t4C] thymidine; DMSO, dimethyl rulfoxida; HAT medium, culture medium containing 10” M hypoxanthine, 10e6 M nminopterin and 5 - lo* M thymidine; HGPRT, hjpoxanthine-rruanine phosphoribosyl- transfera ; MNNG, 1‘.methyl-N’-nitro-N-nitrosoguanidine; gourea; PBS, isotonic saline buffered to pH 7.2 with 0.01 M tocin ; TCA. trichlaroacotic acid.

NMU, NImethyl-N-nit& phosphate; SZ, streptoxo-

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SZ itself is carcinogenic and has been shown to produce mutations in bacte- ria [S] and chromosome aberrations in mammalian cells [5] .

Structural analogs of SZ have been prepared with a view to obtaining an agent which possesses a higher chemotherapeutic ratio relative to SZ [1,2] .

The present work is a comparative study of mutation and DNA damage produced in V79 Chinese hamster cells by SZ, three of its analogs, and MNNG.

MATERIALS AND METHODS

Chemicals SZ and its analogs were prepared by the Upjohn Company. SZ, U-28,601

and U-29,632 were dissolved in 0.01 M potassium phosphate adjusted to pH 4.5. Streptozotocin tetraacetate was dissolved in DMSO. MNNG was dis- solved in acetone. In all cases the solvent used to dissolve the compound was added (20 ~1/5 ml of medium) to the control dishes. SZ is the %deoxy-D- glucose derivative of NMU [l] ; U-28,601 is 3-O-D-glucopyranosyl-NMU [l] ; SZ tetraacetate is SZ with all 4 hydroxyl groups acetylated [l] ; U-29,632 is 3j3-D-galactopyranosyl-NMU [l] .

Cell culture and mutagenesis studies The Chinese hamster cell line (V79), obtained from Dr. E.H.Y. Chu, was

cultured in Dulbecco’s medium, without phenol red, supplemented with 10% fetal calf serum (GIBCO, Grand Island, N.Y .).

We used the protocol of Chu [7] to determine the production of 8-AzG- resistant clones. For each concentration of the test compound 5 plates were used for cytotoxicity determination and 10 plates were used for determining the production of 8-AzG-resistant clones. Plastic petri dishes (60 and 100 mm, Falcon Plastics, Oxnard, Calif.) were used in all experiments. Cyto- toxicity was measured by plating 200 to 1000 cells in 5 ml of medium per dish. 6 h later, various concentrations of the test compound were added and the dishes were incubated for 7 to 9 days. Then the medium was removed and the colonies were fixed with methanol and stained with Giemsa. To determine the % survival, the number of colonies in treated dishes was com- pared with the untreated controls and the latter normalized to 100% surviv- al.

Production of 8-AzG-resistant clones was measured by plating 2 9 lo4 cells in 5 ml of medium per 60-mm dish. 6 h later, the cells were treated with various concentrations of the test compound. 42 h after the addition of test compound, 8-AzG was added to give a final concentration of 30 I.cg/ml in 0.4% DMSO. DMSO (0.4%) was added to the control plates. The medium in the dishes was replaced every 2-3 days with fresh medium containing 30 E.cg/ml of 8-AzG. The cells were stained lo-12 days after plating and the number of colonies counted. The frequency of 8-AzG-resistant cloneswas cal- culated per lo5 survivors, taking into account the number of cells plated and the toxicity.

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Alkaline sucrose sedimentation analysis The production and persistence of single-strand breaks in the DNA of

cells treated with SZ or MNNG were studied by sedimentation analysis in alka- line sucrose gradients using our adaptation [ 81 of the technique of MacGrath and Williams [ 9) . Cells growing in 60-mm petri dishes received [ 14C] TdR (Radiochemical Laboratories, Amersham, Bucks,, England) at a concentra- tion of 0.2 &X/ml in culture medium supplemented with 10% fetal calf serum. After 24 h the radioactive medium was aspirated, the cells were rinsed and incubated with fresh medium for 90 min, after which time an acetone solution of MNNG or solution of SZ in DMSO was added to the culture. The cultures treated with MNNG or SZ were incubated at 37°C for 2 h after which time they were harvested for sedimentation analysis.

The cells were harvested by scraping them into 0.4 mi of ice-cold PBS containing 0.01 M EDTA (pH 7.2). The cells were counted in a hemocyto- meter, and 0.5-1.0 - 10" cells in 0.2 ml of PBS-EDTA was applied to a layer of 0.2 ml of 1 N sodium hydroxide solution on the surface of 5-25% gradients (30 ml) of nuclease-free sucrose (Schwartz-Mann, Orangeburg, N.Y.) containing 0.7 M sodium chloride, 0.3 M sodium hydroxide, 0.01 M Tris, 0.003 M EDTA and 0.06 M p-aminosalicylic acid, adjusted to pH 13.0.

The cells were allowed to lyse for 45 min, and the gradients were centri- fuged at 24 000 rpm for 3 h in the SBllO rotor of a B60 ultracentrifuge (International Equipment Company, Needham, Mass.). Fractions (1.0 ml) were syphoned from the bottom of the gradients and, after receiving 100 c(g of bovine serum albumin, they were treated with ice-cold TCA (12%). The resultant precipitates were collected on glass fiber filter discs (Ge1m.m Type E), washed twice with 4-ml aliquots of ice-cold TCA (2%), dried under infrared lamps, and immersed in toluene-PPO solution for estimation of radioactivity by scintillation counting.

The mean distance through which DNA sedimented in the alkaline sucrose gradients was obtained from the centroid of the sedimentation profile which is calculated according to the following formula: Centroid = Cnifi/Cn, where ni is the net level of radioactivity in the ith fraction fti Control DNA sediments faster and has a higher centroid value than DNA which has been damaged by SZ or MNNG. Sedimentation coefficients were calculated as described previ- ously [ 81 using DNA from T4 bacteriophage as a standard.

RESULTS

Cytotoxicity and mutation The dose-response curve for cytotoxicity (Fig. 1 j shows that MNNG

(IDso = 0.6 pg/ml) is about 600.fold more toxic than SZ (IDS0 = 370 Erg/ml), and SZ tetraacetate (IDS0 = 33 pg/ml) is about lo-fold more toxic than SZ. The SZ analogs: U-28,601 and U-29,632 (IDS0 = 130 pg/ml), are about twice as toxic as SZ. A shoulder can clearly be seen in the survival curve ob- tained with SZ tetraacetate, and small shoulders also seem to be present in the survival curves obtained with the other analogs of SZ; however, SZ itself

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DRUG CONCENTRATION o&ml)

OmmecM 08 12 16 20 c I

Fig. 1. The toxicity of SZ (o), U-29,632 (A) and U-28,601 (A), SZ tetraacetate (X ), and MNNG (a) to V79 Chinese hamster cells. Standard errors range from 5-20% of the data points.

Fig. 2. The relationship between toxicity and the frequency of occurrence of 8-AzG- resistant colonies from V79 Chinese hamster cells treated with various doses of SZ (oj, U-29,632 (a), U-28,601 (A), SZ tetraacetate (x) and MNNG (e). The curve is fitted by eye.

and MNNG did not produce survival curves with clearly evident shoulders. The production of SAzG-resistant clones by the above compounds is

compared in Fig. 2. The results show that at equivalent cytotoxicity similar numbers of resistant clones are produced, indicating that these nitroso com- pounds share a common mechanism for the production of 8-AzG-resistant V79 cells under our experimental conditions.

The frequencies of 6-AzG-resistant clones found in cultures treated with MNNG, SZ or SZ analogs were up to S-fold higher than the frequencies found in control cultures before correction of the data for cytotoxicity. Furthermore, of 10 8-AzG-resistant clones isolated from cultures treated with 2.5 pg of MNNG per ml, none grew in HAT medium. These findings suggest that the 8-AzG-resistant colonies that are represented by the data in Fig. 2 had grown from single cells containing mutations which affect the activity of HGPRT.

Alkaline sucrose sedimentation analysis In the sedimentation profile shown in Fig. 3, it can be seen that a propor-

tion of the total DNA sedimented rapidly to the bcJttom three fractions of the gradient, while a further portion of the DNA remained in the top three fractions of the gradient. However, more than 70% of the total DNA from the control cells sedimented in a broad band having a peak sedimentation value of 140s. By contrast, material from V79 cells containing incorporated

176

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Fig. 3. Alkaline sucrose sedimentation profiles of DNA from V79 Chinese hamster celis treated for 2 h with 250 “g (8), 500 fig (?L) and 1000 pg (e) of SZ/ml. x , Control.

Fig. 4. The relationship betweev toxicity and the centroid of the sedimentation profile DNA from V79 Chinese hamster cells treated for 2 h with various doses of MNNG (O-----+) and SZ (0. - - - - -0). The lines are fitted by regression analysis using a Wang model 600 programmable calculator.

[ 14C] choline and [ “Cl leucine sedimented at 60s and about 5s respectively (Peterson and Heidelberger, unpublished data) demonstrating that the 140s band of DNA does not sediment as an aggregate of lipid and protein. Fur- thermore, DNA from bacteriophage T4 added to the cells in the lysis layer sedimented in a narrow band with a sedimentation constant of 73S, showing that the TJ DNA did not become entangled with the cellular DNA. Thus, the sedimentation behavior of DNA from V79 cells in our experimental system, closely resembles the sedimentation behavior of DNA from lOTI/Z mouse fibroblasts on which we have reported in detail [8] .

Fig. 3 gives the results from a single representative experiment in which treatment of V79 cells with increasing doses of SZ resulted in a dose-dependent diminution of the rate of sedimentation of the DNA, which normally sedi- ments at 140s. Similar results were obtained by treatment of the cells for 2 h with MNNG in a range of doses which produce levels of cytotoxicity com- parabie with those produced by SZ. These results are summarized in Fig. 4.

Fig. 4 shows that the surviving fraction and the centroid of the molecular weight distribution of the DNA decreased on treatment of the cells with MNNC and SZ. For MNNG the centroid changed as a power function of the log of the surviving fraction (correlation coefficient 0.92). A similar rela- tionship between centroid and surviving fraction was suggested by the results obtained with SZ (Fig, 4), although a meaningful correlation coef- ficient was not obtained due to the variability associated with replicate analyses with this compound. The variability is adequately explained by the instability of SZ in storage as a dry powder at 4°C [ 3 ] .

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DISCUSSION

Recent studies in several laboratories have shown that the growth of cells in medium containing 8-AzG is governed by a complex interreiationship of many factors, although resistance often stems from a genotypic deficiency of the enzyme HGPRT [7,10-121. However, the HAT sensitivity of the 8-AzG-resistant clones produced in the’course of the present study, together with our previous detailed analysis of reversion of HAT sensitive, &AzG-re- sistant V79 cells [ 111, suggest that the results shown in Fig. 2 reflect induc- tion by MNNG and by SZ analogs of mutations (alterations in the primary structure of the DNA) in the treated V79 Chinese hamster cells.

We therefore consider that Fig. 2 shows an association between the pro- cesses of mutation and cytotoxicity in V79 cells treated with MNNG and SZ and its analogs. Other laboratories have also obtained results that suggest associations between mutagenicity and cytotoxicity induced in a variety of mammalian cells by alkylating agents [ 13,141, polycyclic hydrocarbons [15] and ultraviolet light [16] .

The high mutation rates commonly found in studies of mammalian as com- pared with bacterial cells [ 7;i2] have led one group of workers to put forward the theory that the primary damage induced in mammalian cells by mutagens may not be located within the DNA [16] . However, our data show that the phenomena of’ DNA damage and mutation are associated processes in V79 Chinese hamster cells treated with MNNG or SZ in the dose range LD40 -LDso, since the incidences of both phenomena increase as a function of toxicity (Figs. 2 and 4).

The relationships obtained between DNA damage and toxicity with cells treated with1 MNNG in the present study (Fig. 4), are consistent with our previous findings which showed that the centroids of the sedimentation profiles of DNA from mouse fibroblasts treated with MNNG decreased as a power function of dose [8,17]. Furthermore, we have found that the DNA damage measured by our sucrose gradient procedure represents single-strand breaks produced at methylated sites on the DNA of cells treated with MNNG [8] , and this finding is consistent with the results obtained by others [ 181. Thus, since both MNNG and SZ possess methyl nitroso groups, the data in Fig. 4 suggest that doses of MNNG and SZ which are equal- ly toxic to V79 cells, methylate the DNA of these cells to the same ex- tent

In conclusion, we interpret the results obtained in the present study to suggest that widely disparate levels of MNNG and SZ, and by inference the SZ analogs that we have tested, produce in V79 Chinese hamster cells a uniform ass’ociation among the processes of cytotoxicity , mutation and DNA methyllation.

ACKNOWLEDlGEMENTS

We thank Ms. Cathy Cita, Ms. A. Macaluso and Ms. H. Peterson for skilled technical assistance, Mr. D. Krahn for helpful discussions, and Dr. H. Camp- bell for help with the mathematical analyses.

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