Chem. Res. Toxicol. 1989,2, 157-161
157
Intracellular Activation of Cytotoxic Agents: Kinetic Models for Methylnitrosoureas and N-Methyl-N’-nitro-N-nitrosoguanidine in Cell Culture Robert J. Weinkam” and M. Eileen Dolant Drug Metabolism Department, Allergan, Inc., 2525 Dupont, Irvine, California 92715, and Department of Medicinal Chemistry and Pharmacognosy, Purdue University, W e s t Lafayette, Indiana 47907 Received November 28, 1988 T h e cytotoxic activity of N-methyl-N-nitrosourea (MNU), streptozotocin, and N-methyl-
N’-nitro-N-nitrosoguanidine(MNNG) was determined in cell culture by using a P388 cell growth rate inhibition assay. These agents appear t o have very different activities when inhibition is related to the agent concentration in the culture medium: ED,(CO) = 40 pM for MNNG t o 875 pM for streptozotocin. T h e mechanism of action of these three agents involves conversion t o the active methanediazonium ion and subsequent methylation of cellular macromolecules. As a consequence, the rates of conversion of the parent agent to the methylating species in the medium and within the cell are important parameters that also need to be considered t o reach a more detailed understanding of the mechanism of action. In order to do this, a kinetic model has been developed to calculate the concentration of drug that is converted to active methylating species within the cell during the assay incubation period. The use of cell culture kinetic models was extended from simple compounds activated through solvolytic reactions (nitrosoureas) t o a n agent that undergoes selective intracellular activation (MNNG). By use of measured values for initial drug concentration, incubation time, and cell volume, as well as extracellular and intracellular chemical activation rate constants, the intracellular concentration, [P4],which represents the cumulative intracellular reaction products formed during the incubation period, between 140 was calculated and related t o cytotoxicity. All three agents showed an EDb0[P4] and 180 pM, and for MNNG, this ED, was independent of extracellular sulfhydryl concentration. These data are consistent with a mechanisms in which the observed cytotoxicity is proportional to the amount of active methanediazonium ion, and resulting reaction products, that is generated within the cell and independent of the structure of the methanediazonium ion precursor. The data support the use of cell culture kinetic functions as a means to further understand the relation between drug dose and observed activity.
Cells in culture can be used in place of animal or tissue preparations to investigate the activity of drugs and toxic substances. The interpretation of data obtained from culture assays run under different conditions and between drugs that act through different mechanisms is a continuing problem as is the obvious difficulty of extrapolating data from cell culture to whole tissues and living animals. The controlled parameters of the cell culture experiment (dose, incubation time, temperature, cell density, and the addition of activation or inhibitory factors to cells or media) provide an opportunity to develop an understanding of the kinetic and mechanistic characteristics of the drug in the culture system that can aid in data interpretation and extrapolation to other systems. This paper describes cell culture cytotoxicity experiments in which the mechanism of drug action and cell culture kinetic models is used in data analyses and interpretation. Three carcinogenic and cytotoxic methylating agents that act through the same ultimate active species, the methanediazonium ion, were studied. NMethyl-N-nitrosourea (MNU)l and streptozotocin, a 2deoxyglucose-substituted N-methyl-N-nitrosourea, are
agents that hydrolyze a t different rates through generalbase catalysis to give the methanediazonium ion (1, 2). This species reacts with cellular macromolecules including DNA (3-5) to cause cell toxicity, mutations, and carcinogenicity (3,6, 7). Methylnitrosourea hydrolysis also generates an isocyanate that can react with amines to generate stable ureas (3). MNNG is a potent mutagen and carcinogen known to produce gastrointestinal tumors in laboratory animals (8-1 1). Similar to methylnitrosourea, this agent appears to act through a reactive intermediate, the methanediazonium ion, which can covalently bind to cellular macromolecules (12,13). Products formed from the reaction of this active species with bases of DNA are likely to be critical lesions in the initiation of tumors (14). MNNG decomposition also generates a guanidinating group which reacts with lysine of basic proteins such as histones or cytochromes to form nitrohomoarginine residues (15-18). The methylating agent MNNG is of particular interest due to the unique chemistry involved in its reaction to form an active alkylating intermediate. In neutral aqueous solution, it decomposes to yield a methylating species and
* Address correspondence to this author a t t h e Drug Metabolism Department, Allergan, Inc., 2525 Dupont, Irvine, CA 92715. Present address: Department of Physiology and Cancer Research Center, The Milton s. Hershey Medical Center, The Pennsylvania State University College of Medicine, Hershey, PA 17033.
Abbreviations: MNU, N-methyl-N-nitrosourea; MNNG, N methyl-N’-nitro-N-nitrosoguanidine; ENNG, N-ethyl-N’-nitro-Nnitrosoguanidine; ED,(C,) or EDw[P4],dose required to cause a 50% reduction in growth rate measured as initial drug concentration, Co, or intracellular alkylation product [P,]; GSH, glutathione; PBS, phosphate-buffered saline.
0893-228x/89/2702-0157$01.50/0 0 1989 American Chemical Society
Weinkam and Dolan
158 Chem. Res. Toxicol., Vol. 2, No. 3, 1989 Scheme I NH N=O
I
CH3N=NOH
+
II
+ RSCNHNO,
C H ~ ~ C N H N O , RSH .
I1 NH
\
CH,NHCNHNO,
II NH
+ RSNO
a salt of nitrocyanamide (12). More importantly, the formation of the active methylating intermediate is greatly accelerated in the presence of sulfur nucleophiles (12,19, 20). The sulfur atom reacts covalently with the amino carbon of MNNG and releases the reactive methylnitrosamine group. There is also attack at the nitroso group of nitrogen, which leads to denitrosation and formation of N-methyl-N-nitroguanidine(Scheme I). Reports indicate that the incubation of MNNG with DNA results in more extensive alkylation of DNA if cysteine is present (12, 20). In contrast to the nitrosoureas, the presence of intracellular thiol-containing compounds, such as glutathione, might significantly alter the rate and extent of alkylation caused by these agents. The properties of these reactions are important in the development of a kinetic model that represents the amount of active species formed in the cell. Previous studies developed a model for (chloroethy1)nitrosoureas (21,22), which is extended in this report to an agent, MNNG, that undergoes selective intracellular activation.
Materials and Methods Chemicals. N-Methyl-N'-nitro-N-nitrosoguanidine and Nethyl-N'-nitro-N-nitrosoguanidine were obtained from Aldrich Chemical Co. (Milwaukee, WI) and stored a t 5 "C. N-MethylN-nitrosourea, streptozotocin, glutathione in the reduced form, and all other biochemicals were purchased from Sigma Chemical Co. (St. Louis, MO). For these same studies, MNNG was prepared immediately before use by dissolving in ethanol and then diluting to the desired volume with distilled water. For MNNG decomposition, the internal standard ENNG was dissolved in methanol and kept on ice. Cytotoxicity Studies. P388 mouse leukemia cells were maintained in suspension culture with Fisher's growth medium supplemented with 10% (v/v) donor horse serum, 100 pg/mL streptomycin, and 100 units/mL penicillin G. Stock cultures were grown in stationary bottles a t 37 "C, under 5% COz/humidified air. Cells were diluted every 3-4 days with fresh medium to attain a final concentration of 5 X lo4 cells/mL. Approximately 24 h prior to treatment, cells were planted in a 250-mL spinner flask a t a density of 3 x lo5 cells/mL. The cytotoxicity experiments have been described in detail previously (22). Briefly, two different types of experiments were performed. The initial concentration of drug was varied by using a defined incubation period, or the exposure period was varied during which a defined initial drug concentration was incubated with cells a t 37 "C and p H 7.4. In the first set, drug was added in increasing amounts to a series of tubes containing 2.0 mL of cells. After the exposure period, 0.2-mL aliquots were added to 3.8 mL of fresh media. Cells were incubated a t 37 "C for 48 h. In the second set of experiments, a known amount of drug was added to 18.9 mL of cells and 0.27-mL aliquots were added to 3.8 mL of fresh media a t various time periods. The growth fraction (F)represents the growth rate ratio of treated relative to nontreated cells. Similar experiments were also performed by using phosphate-buffered saline (PBS) or Fisher's supplemented with 18 units of acetylcholinesterase (Sigma) in place of Fisher's growth medium for the period of incubation of cells with drug. Rate of MNNG Decomposition. The rate of disappearance of parent drug MNNG from solution was determined by using high-pressure liquid chromatography. N-Ethyl-N'-nitro-Nnitrosoguanidine (ENNG) dissolved in methanol was used as the
internal standard. Chromatographic analysis was carried out by using an Altex C185-pm Ultrasphere reverse-phase column (Altex Scientific Co., Berkeley, CA). The mobile phase consisted of 25% Chrom Ar grade methanol/75% doubly distilled water. MNNG and ENNG were separated isocratically by using 25% methanol/water a t a flow rate of 1.5 mL/min and monitored by UV analysis a t a wavelength of 270 nm. A stock solution of MNNG in methanol was added to preheated (37 "C) Fisher's growth medium or 0.025 M PBS to give a final concentration between 20 and 48 pmol. Solutions were kept a t p H 7.4 and 37 "C. Experiments were performed in medium without cells and with (1.0-7.0) X lo6 cells/mL. A 100-pL aliquot was removed a t various time periods and centrifuged a t 15600g to remove extraneous cell material. An equivalent amount of ENNG, the internal standard, was added to the tubes to give a final concentration of 13 pM. From this final solution, 20 pL was injected onto the column. The disappearance of MNNG was calculated from the peak height ratio by reference to a standard MNNG/ENNG curve. Cell C u l t u r e Kinetic Model. The kinetic model described below is applicable to compounds that act through the irreversible formation of intracellular reaction products. Rate constants can be measured or estimated and used to calculate the amount of intracellular reaction product formed. This quantity, rather than the initial concentration of the agent in the medium, can be related to observed cellular effects. The model used for MNU, streptozotocin, and MNNG is shown in eq 1. Terms A, and Pz are extracellular amounts of agent and *12_ A1
p2 (1)
Ag
P,
reaction products, respectively. Intracellular amounts are represented by A, and Pk The intermediate methanediazonium ion is not represented in this equation as it has a transitory existence that is kinetically indistinguishable from the direct formation of products from parent drug. Some simplifying assumptions can be applied. Most small molecules distribute rapidly between medium and suspended cells (23) so that equilibrium is assumed to occur before significant amounts of A, react in the medium. The free aqueous concentration of drug in the cell is also assumed to equal that in medium a t equilibrium (k13 = k,, >> klz). The concentrations [A,] and [A3]a t time zero are considered to equal the initial concentration of drug in medium, Co. Drug that partitions into cell lipid is ignored as it is a small fraction of the total and drug degradation is not believed to occur a t an appreciable rate in the lipid matrix (24). The rate laws for this model are shown in eq 2-5. T h e cumulative concentration of [P4] between initiation of the drug
[A,], = Coe-(k12+kaVc)t
[P4], = [A,],(l - &zt)
(2)
(5)
exposure, to,and termination, t , is given in eq 4, where V , is the ratio of cell volume per milliliter of medium. The P, term represents all cellular reaction products resulting from the active methylating species. A small fraction of these products may be altered macromolecules that lead to observed changes in growth rate. Equation 4 can be simplified further if the activation rates in medium and the cell are equal, k12 = k34, and the fractional cell volume is small, to give eq 5. The latter equation is applicable to alkylnitrosoureas (21, 22).
Results The cell culture assay system used in these studies measured drug-induced inhibition of P388 mouse leukemia
Chem. Res. Toxicol., Vol. 2, No. 3, 1989 159
Cytotoxicity Kinetic Model
1 .O
0.6
F 0.4
V
~~
200
400
600
800
1000
I
I
I
I
I
10
20
30
40
50
c o (PM)
TIME (min)
Figure 1. MNU (o), streptozotocin (A),and MNNG (0) P388
cell growth rate inhibition (F)is plotted against initial drug concentration (C,) for respective incubation times of 30,45, and 15 min. MNNG is active at lower concentrations than the methylnitrosoureas in spite of the shorter incubation period.
Figure 3. The growth rate inhibition (F)for 7.5 pM MNNG in PBS ( 0 )and Fisher's medium containing 10% donor horse serum (0) is plotted against incubation time.
1.01
0 100
200
300
400
IP41 (PM)
Figure 2. Calculated values of intracellular reaction product [P4] are plotted against P388 cell growth rate inhibition (F)for MNU
(a),streptozotocin (o), and MNNG (0). The three curves are the same within experimental error.
cell population growth rate in suspension culture relative to untreated controls. Experiments were conducted at a cell density of lo6 cells/mL, pH 7.4, and 37 "C with either the initial concentration, [A,], = Co, or incubation time, t , varied. Figure 1 shows the effect of incubating MNU, streptozotocin, and MNNG with exponentially growing P388 cells. Results are expressed as growth fraction (F = 1- treated/control) against initial agent concentration (C,). MNNG, EDm(CO)= 4.1 f 0.5 pM, t = 15 min, is active at much lower concentrations and at a shorter incubation time than MNU, EDm(Co)= 227 f 15 pM, t = 30 min, and streptozotocin, EDN(CO)= 830 f 70 pM, t = 45 min. MNU and streptozotocin are known to react in solution to give methylation products in high yield (1,2). The rate constants for reaction under cell culture incubation condition are klz = 0.087 min-' and 0.003 min-' for MNU and streptozotocin, respectively (1,24). Since the half-life of streptozotocin is 230 min, it is apparent that at least part of the low observed activity is due to incomplete conversion to the active methylating species during the 45-min incubation period. A more quantitative interpretation can be made by relating activity to the total cellular reaction product, [P4], calculated for the respective incubation periods. Since nitrosourea conversion to give methylation products is not catalyzed by thiols (20), [P4]may be calculated by using the simplified eq 5. Use of this equation permits data obtained under different conditions (C,and t ) to be averaged and compared in Figure 2. When plotted in this
1
2
3
4
5
6
CELL NUMBER (10' cells/ml)
Figure 4. MNNG disappearance rate constants (k)in PBS ( 0 ) and Fisher's medium (0) are plotted against P388 cell number. The y-axis intercept is the rate constant in the absence of cells. The intracellular rate constant is obtained from the slopes. Both curves have a slope of 0.0021 mL/ ( lo6 cellpmin).
way, the two curves are the same within experimental error. The behavior of MNNG in this assay is more complex than that of the nitrosoureas. MNNG appears to be much more active than MNU in Figure 1. The activity, however, is affected by the cell culture medium. In Figure 3, the activity of 7.5 pM MNNG over time against cells in phosphate-buffered saline (PBS) appears to be greater than that against cells in Fisher's medium containing 10% (v/v) donor horse serum. In order to model the kinetic behavior of MNNG, it is necessary to determine the rate constants for extracellular and intracellular chemical activation. The rate of MNNG (20-40 pM) disappearance from 0.025 M PBS and Fisher's medium containing 10% serum was determined at 37 "C and pH 7.4 by using reverse-phase HPLC. Extracellular rate constants in PBS and medium were 0.0048 min-' and 0.0197 min-', respectively, with the faster rate due to the presence of sulfhydryl compounds in the serum-containing medium. The intracellular rate constant was determined by incubating MNNG with Fisher's medium and PBS containing (1.0-7.0) X lo6 cells/mL (Figure 4). The rate constant for reaction of MNNG in P388 cells suspended in either medium was 0.0021 min-'/(106 cellsmml). The intracellular half-life of MNNG is 6.8 s. The slopes of both curves are the same, indicating that MNNG reacts at the same intracellular rate independent of small differences in extracellular sulfhydryl concentration. Since MNNG methylation reactions occur in 50% yield (12,20,25),the rate constant for formation of methylation
o*6rmDm/~
160 Chem. Res. Toxicol., Vol. 2, No. 3, 1989
0.8
$2mM GSH
F 0.4
p”
20
40
Weinkam and Dolan
jacent guanine (34). The data of this report are not consistent with the occurrence of specific interactions between macromolecules and the parent drug structure that would lead to a localized distribution of alkylation damage. The relationship between cell toxicity and [P4] may apply to all methylation agents that act through the methanediazonium ion. The data of Figure 2 can be expressed as eq 6 to relate the growth fraction F to the [P4] term. By combination of eq 5 and 6, the growth fraction can be predicted for streptozotocin and methylnitrosourea initial concentrations or incubation periods.
F = (4 X 10-3)[P4]- (4.6 X 10+)[P4l2 60
SO
100
c o (PM)
Figure 5. The growth rate inhibition (F)for MNNG is shown vs initial concentration (C,) for drug in Fisher’s medium and Fisher’s medium containing 2 mM added GSH.
products within the cell is kS = 3.05 m i d . The P388 cells employed in these studies had a mean diameter of 8.7 X lo4 m (lo6cells have a volume of 3.45 X lo4 mL) and an average sulfhydryl content of approximately 20 nmol/ lo6 cells, measured by using the Ellman reagent. The value for [P4]can be calculated by substituting these parameters into eq 4. Plotting activity, F , against [P4]gives a curve, Figure 2, that is also the same as that observed for MNU and streptozotocin. An additional experiment was conducted to confirm that activity difference between media was related to extracellular sulfhydryl concentration. Figure 5 shows results from an experiment in which graded concentrations of MNNG are incubated with cells for 15 min in medium containing 2 mM reduced glutathione. The apparent activity of MNNG was reduced by extracellular sulfhydryl. The ED50(Co)of 78 pM approaches that of MNU.
Discussion The cell culture cytotoxicity of the nitrosoureas MNU and streptozotocin is shown to be equal when referenced to the amount of methylation product formed within the cell during the assay incubation period. A similar observation was made with (chloroethy1)nitrosoureaderivatives by using the same P388 cell growth rate assay (22) or 9L cell colony forming efficiency assay (21). When the cytotoxic activity is related to the amount of intracellular methylation [P4],the ED50 values for MNU and streptozotocin are between 140 and 180 pM. According to the same assay, the ED,[P,] values for several (chloroethy1)nitrosoureas are between 6 and 7 pM (22). The observation that members of a methyl- or (chloroethy1)nitrosourea series have the same cytotoxic activity is consistent with a mechanism of action in which each series is a precursor to a common alkylating intermediate that is randomly generated within the cell. The fact that (chloroethy1)nitrosoureas have a 25-fold lower ED5,[P4] than methylnitrosoureas may indicate that 2-chloroethylation of macromolecules is more efficient or damaging to cell growth than methylation. Sequence-preferred alkylation of guanidines has been reported for MNU (26, 27) and also for (chloroethy1)nitrosoureas (28, 291, alkyltriazines (30), and nitrogen mustards (31). Methylation of dodecamers by MNU occurs preferentially, 1.5- to 3-fold, on the N7- or 06-position of guanines preceded at the 5‘ end by a purine rather than a pyrimidine (27). This result has been explained by the influence of electrostatic forces (32) by base stackng that affects steric accessibility (33),and by an MNU-purine reaction intermediate that directs methylation to an ad-
(6)
MNNG shows growth rate inhibition effects at much lower concentrations than MNU or streptozotocin. This fact can be related to the differences in reaction rates for MNNG activation in medium and in the cell. In an aqueous environment, MNNG reacts through a base-catalyzed first-order reaction to form the active methylating intermediate (9). In PBS at 37 “C,pH 7.4, this is a relatively slow reaction, k12= 0.0048 min-l, tl12= 147 min. In the presence of thiols, such as glutathione and cysteine, the rate of MNNG decomposition is greatly accelerated (12,19,20). The measured sulfhydryl concentration of the P388 cells used in this study was 50 mM, which produced a much more rapid rate of MNNG activation within the cytosol, k34 = 6.1 min-l, tllz = 6.8 s. As a consequence, there is a preferential formation of active species within the cell and increased activity relative to the initial concentration of MNNG in the medium. These factors can be treated quantitatively to calculate the amount of intracellular alkylation product [P4]by using eq 4 and measured rate constants. When MNNG activity is plotted against [P4], the activity is the same as that of the methylnitrosoureas (Figure 2 ) . This result is consistent with a mechanism in which the cytotoxic activity of MNNG is dependent on the amount of alkylated product formed within the cell during the exposure period. The potent cytotoxicity of MNNG has been attributed to reactions of the guanidinating group produced from MNNG decomposition with cellular molecules, particularly proteins and histones, along with alkylation of nucleic acids (17,35-38). The data presented in this paper suggest that the high cytotoxicity of MNNG is a function of the kinetics involved in the formation of the active methylating species rather than the guanidination reaction. The utility of the kinetic model shown in eq 1 in understanding cell culture cytotoxicity is also shown in the analysis of the apparent differences in activity seen when cells are incubated in different media (Figure 3). In this case, lower MNNG activity is seen in Fisher’s medium with 10% horse serum. This is shown to be related to the higher rate of apparently nonproductive degradation of MNNG in that medium, which decreases the amount of drug available to react within the cell. The activity of MNNG, when compared to [P4], is the same in both media, as is the intracellular reaction rate constant. Future studies will extend the use of kinetic models to agents that are metabolically activated within cells to direct acting irreversible inhibitors. Use of cell culture kinetic models can provide new insight into factors that determine activity in culture and may prove to be an important tool in the analysis and interpretation of cell culture activity assays. Acknowledgment. This work was supported by the National Cancer Institute, Grant CA-26381. Cell culture studies were supported by the Purdue University Cancer Center, CA-23168.
Cytotoxicity Kinetic Model Registry No. MNU, 684-93-5; MNNG, 70-25-7; streptozotocin, 18883-66-4.
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