A Kinetic Model of the Cell Culture Cytotoxicity of Metabolically

much different from drug levels in the medium. A kinetic approach to the analysis of cell culture activity data was described in previous reports for ...
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Chem. Res. Toxicol. 1989, 2, 288-294

288

A Kinetic Model of the Cell Culture Cytotoxicity of Metabolically Activated Agents: N-Methyl-N-(acetoxymethyl)nitrosamine, Methylnitrosourethane, and (Met hy1azoxy)methanol Acetate Robert J. Weinkam* and Irene Plakunovt Drug Metabolism Department, Allergan, Inc., 2525 Dupont, Irvine, California 92715,and Department of Medicinal Chemistry and Pharmacognosy, Purdue University, West Lafayette, Indiana 47907 Received January 13, 1989 The activity of three metabolically activated methylating agents, N-methyl-N-(acetoxymethy1)nitrosamine (DMN-OAc), methylnitrosourethane (MNUT), and (methy1azoxy)methanol acetate (MAM-Ac), were determined in cell culture by using a P388 cell growth rate inhibition assay. Experiments were conducted with normal P388 cells in Fischer’s medium and under conditions in which the esterase-mediated activation was modified by pretreating cells with the irreversible esterase inhibitor paraoxon and by adding acetylcholinesterase t o the medium. Inhibition of intracellular esterase had a much greater effect on activity than did addition of enzyme to medium. These experiments provided data t h a t were used to assess the utility of kinetic models as a means to gain a more detailed understanding of the cytotoxicity process in cell culture. Growth rate inhibition was related to the amount of intracellular alkylation resulting from formation of metabolic intermediates and their subsequent chemical reaction to form methyldiazonium ion and methylation products by using kinetic rate laws and measured rate constants. T h e model is applicable to systems that form unstable metabolites that can, in part, partition between the cell volume and incubation medium. When growth rate inhibition effects were related to cumulative intracellular alkylation [PI, the ED,, values were the same for all three agents and for three previously reported chemically activated methylating agents, Nmethyl-N-nitrosourea, streptozotocin, and N-methyl-N’-nitro-N-nitrosoguanidine, which are also thought to act through the methyldiazonium ion. This observation is consistent with a growth rate inhibition effect of the methyldiazonium ion that reflects the intrinsic activity of this species that is independent of the precursor molecule. Extracellular activation of DMN-OAc and MNUT were shown to be much less efficient in causing growth rate inhibition than intracellular activation for these agents that act through metabolites with half-lives of 20-40 s. The activity of MAM-Ac was not consistent with a model t h a t used the reported rate constant of the stable MAM metabolite but could be explained by a mechanism that involved a rapid intracellular activation of MAM to the methyldiazonium ion.

Introduction Cell culture systems are now commonly used to measure biological activity, cytotoxicity, and mutagenicity. In most assays, biological activity is related to the initial concentration of drug in the culture medium. This relationship may not be appropriate, however, for compounds that undergo transformation during the assay period. Further complications may arise for compounds that are irreversible inhibitors or that act through reactions, such as alkylation of cell components, as these agents may accumulate within the cell at local concentrations that may be much different from drug levels in the medium. A kinetic approach to the analysis of cell culture activity data was described in previous reports for chemically activated alkylating agents (1,2) and for compounds that undergo preferential intracellular activation (3). Measured rate constants and kinetic rate laws were used to correlate cell culture assay data with calculated concentrations of intracellular reaction products. This kinetic approach has been extended in this paper to include agents that undergo *Address correspondence to this author at the Drug Metabolism Department, Allergan, Inc., 2525 Dupont, Irvine, CA 92713. ‘Present address: Bausch & Lomb, Inc., 1400 North Goodman St., Rochester, NY 14692.

metabolic activation to intermediates that act through intracellular alkylation. Growth rate inhibition data have been obtained on three compounds, N-methyl-N-(acetoxymethy1)nitrosamine (DMN-OAc)’ (4,5), methylnitrosourethane (MNUT) (6), and (methy1azoxy)methanol acetate (MAM-Ac) (7). Each of these agents undergoes hydrolysis of an ester function to form a metabolic intermediate, which reacts chemically to generate a common active methylating species, the methyldiazonium ion. DMN-OAc and MAM-Ac hydrolyze to form intermediates that are the same as oxidative products of dimethylnitrosamine (8) and 1,2-dimethylhydrazine (9),respectively. The structures and activation pathways are shown in Figure 1. Earlier studies with methylnitrosourea (MNU), streptozotocin, and N-methyl-N’-nitro-N-nitrosoguanidine Abbreviations: MNU, N-methyl-N-nitrosourea; MNNG, N methyl-N’-nitro-N-nitrosoguanidine; ENNG, N-ethyl-N’-nitro-Nnitrosoguanidine;DMN-OAc, N-methyl-N-(acetoxymethyl)nitrosamine; DMN-OH,N-methyl-N-(hydroxymethy1)nitrosamine; MNUT, methylnitrosourethane; MAM-Ac, (methylazoxy)methanolacetatq MAM, ( m e thy1azoxy)methanol;ED,(C,) or ED,(P), dose required to cause a 50% reduction in growth rate measured aa initial drug concentration C, or intracellular alkylation product [PI;AcChE, acetylcholinesterase; PBS, phosphate-buffered saline; V,,volume of 1 x 10s P388 cells/mL of medium.

0 1989 American Chemical Society

Chem. Res. Toxicol., Vol. 2, No. 5, 1989 289

Metabolically Activated Methylating Agents

+

0

/O N

0

11

I

II

CyN=NCH@CH,

c yL b C x C Y

Methylaoxymetharw Acetate

N-methyl-N-acatorymethylnnmsamine

CCgN~YCY

a

N-methyl-N.ni~rOSOurethane

MAM AC

DMN.OAc

MNUT

I

1

I

esteraseIH,O

esteraselH,O

do

+

0 CYN-WYOH

k

-

do

I

I

CYW4a-l

Methylazoxymethanol

MAM

esteraselH,O

\

0 OOllmm

CtgNsoC-

N-Methyl-N-hydroxymethylnitrosamine N-Methyt-N.nltro~ccarbam~c

DMl:rl 7"

/

k = 047mn

CyN=N

Figure 1. The hydrolyses of (methy1azoxy)methanol acetate (MAM-Ac), N-methyl-N-(acetoxymethy1)nitrosamine(DMNOAc), and N-methyl-N-nitrosourethane(MNUT) by cellular esterases form metabolic intermediateshaving different stabilities that react to give a common active species, the methyldiazonium ion. (MNNG), which also act through the methyldiazonium ion (10, I l ) , indicated that P388 cell growth rate inhibition could be related to the accumulated amount of methyldiazonium ion that formed and reacted within the cell volume during the assay incubation period (3). This activity, EDSO(P)= 140-180 pM, was the same for all three agents while activity relative to initial drug concentration EDSO(C)differed substantially. The kinetic models used to correlate data for these agents equated formation of intracellular @lation products to disappearance of parent drug in the cell. This model could not be applied to DMN-OAc, for example, as this agent is converted to a metabolic intermediate, N-methyl-N-(hydroxymethy1)nitrosamine (DMN-OH), that may be sufficiently stable, k = 0.41 mi&, to partition between the cell volume and medium (12). A kinetic model that includes terms to describe transcellular partitioning of metabolites has, therefore, been developed to calculate the amount of intracellular alkylation product formed during the assay period. The compounds described are converted, a t different metabolism rates, to metabolic intermediates having a range of stabilities. The model was tested further by modulating the metabolic hydrolysis rate by pretreating cells with the esterase inhibitor paraoxon and by adding acetylcholinesterase (AcChE) to the medium.

Materials and Methods Chemicals. (Methy1azoxy)methanol acetate, l-nitrosopyrrolidine, and N-methyl-N'-nitro-N-nitrosoguanidine were obtained from Aldrich Chemical Co. (Milwaukee, WI). NMethyl-N-nitrosourethanewas from Pfaltz and Bauer (Stamford, CT). Acetylcholinesterase (type 111, EC 3.1.1.7) was purchased from Sigma Chemical Co. (St. Louis, MO), and Fisher's medium for leukemic cells of mice and donor horse serum were from G i b Laboratories (Grand Island, NY). P388 mouse leukemia cells were obtained from EG&G Mason Research Institute (Worchester, MS). Cytotoxicity Studies. P388 mouse leukemia cells were maintained in suspension culture using Fisher's growth medium supplemented with 10% (v/v) donor horse serum, 100 pg/mL streptomycin, and 100 units/mL penicillin G. Stuck cultures were grown in stationary bottles at 37 "C, under 5% C02/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 at a density of 3 X lo6 cells/mL. The cytotoxicity experiments have been described in detail previously (2). Briefly, two different types of experiments were performed, either varying the initial concentration of drug at a

defined exposure time, 30 min, or varying the exposure period during which a defined initial drug concentration was incubated at 37 OC and pH 7.4. In the concentration-varying experiments, four sterile glass tubes containing 2.0 mL of a lo6 cells/mL suspension were prepared for each nontreated control and drug concentration to be tested. Drug solutions were freshly prepared in water or in a minimum volume of ethanol diluted with water immediately prior to use. Small aliquots, less than 100 pL, were added to the cell suspension and vortexed to give final drug concentrationsbetween 0 and 500 pM. All tubes were incubated at 37 OC for 30 min. At the end of the exposure period, 0.2 mL was transferred from each tube to a similar tube containing 3.8 mL of medium. The 20-fold dilution was considered to quench the drug effect. All cell suspensions were incubated at 37 "C for 48 h and the resulting cell populations determined by counting viable cells, as indicated by trypan blue staining, using a hemocytometer. Each tube was counted in duplicate, and the results were averaged. In the time-varying experiments, freshly prepared drug solution was added to 19 mL of a cell suspension, lo6 cells/mL, that was maintained at 37 "C. Aliquots, 0.27 mL, were removed immediately prior to drug addition and at varying times after drug addition. Four aliquots were taken at each time point, and each aliquot was added to a tube containing 3.8 mL of medium. All tubes were then incubated at 37 O C for 48 h and the cell populations determined, as described above. The fractional growth inhibition was determined from the ratio of treated to nontreated control cell populations by using the equation F = 1 - (treated/control). Similar experiments were performed by using phosphatebuffered saline (PBS) instead of Fisher's growth medium for the incubation period of cells to drug. Prior to exposure to drug, cells were centrifuged and resuspended in sterile PBS. Another type of experiment involved the addition of AcChE, 18 units per 0.27-mL aliquot, to the Fisher's growth medium prior to the exposure of cells to drug. Cytotoxicity experiments were also conducted with P388 cells that were pretreated with paraoxon. A paraoxon solution was prepared by transferring 25 pL of liquid (cautionshould be used) via a syringe to a 2-mL volumetric flask. The agent wiw dissolved in 200 pL of ethanol and diluted with the water to final volume. A 1.75-mL aliquot of this solution was added to 100 mL of cells to give a final concentration of 1.01 mM. After exposure for 20 min, 10 mL of cells was centrifuged at lO00g for 10 min. Cells were resuspended in serum-free medium and used in the above cytotoxicity experiments. Rates of Chemical Hydrolysis. The rate of disappearance of parent drug from solution was determined by using high pressure liquid chromatography. 1-Nitrosopyrrolidinedissolved in methanol was used as the internal standard. Chromatographic analysis was carried out by using an Altex CIS5-pm Ultrasphere reverse-phase column (Altex Scientific Co., Berkeley, CA). The mobile phase consisted of Chrom Ar grade methanol/doubly distilled water. The solvent ratios for DMN-OAc, MNUT, and MAM-Ac were 25:75,5050, and 2080, respectively, at a flow rate of 1.0 mL/min, and the effluent was monitored by UV analysis at a wavelength of 230 nm. Solutionsof DMN-OAc (78 pM), MNUT (90pM),and MAMAc (115 pM), were prepared in 0.15 M, pH 7.4, PBS or Fisher's medium containing 10% serum. The solutions contained the 1-nitrosopyrrolidine standard (30 pM). Solutions were kept at pH 7.4 and 37 OC, and 20-pL aliquots were assayed for parent drug over 2 h (MNUT) or over 28 h (DMN-OAcand MAM-Ac). Disappearance was calculated from the peak height ratio by reference to a standard MNNG/ 1-nitrosopyrrolidine curve. Hydrolysis rates were also measured in the presence of P388 cells. A solution of drug and internal standard was added to each of five 10-mL volumetric flasks containing 1 X lo6, 2 X lo6, 4 X lo6, 6 X lo6, and 8 X lo6 cells/mL to give final concentrations of 90 and 40 pM, respectively. Solutions were incubated at 37 "C, and 1-mL aliquots were removed periodically, centrifuged at 15600g for 1 min, and assayed for parent drug. Hydrolysis rates were also measured in the presence of AcChE. Solutions containing 30 units of AcChE (EC 3.1.1.7 Sigma Chemical Co. type 111),0.23 pmol of 1-nitrosopyrrolidine, and 0.125-1.75 pmol of DMN-OAc, MNUT, or MAM-Ac in a 2.0-mL

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Chem. Res. Toxicol., Vol. 2, No. 5, 1989

Weinkam and Plakunov

final volume of 150 mM, pH 7.4, phosphate buffer were held at 37 "C. Reactions were monitored over 1h by HPLC analysis of parent drug. Cell Culture Kinetic Model. The kinetic model of eq 1 is applicable to agents that are converted to metabolites that may A1

03

'

-kl2

M2

kp3

Pa

partition between the cell and surrounding medium. The intermediate is subsequently converted to a product P, which may be related to biological activity as a covalently bound reaction product or an irreversible inhibitor of a cellular process. In this model A,, M2, and P3are extracellular agent, metabolite, and reaction products, respectively, while A4, M5, and P6 are corresponding intracellular amounts. The model was applied to DMN-OAc, MNUT, and MAM-Ac, which were converted, following metabolic or chemical hydrolysis, to the methyldiazonium ion. This very reactive species is not represented in the model as it is kinetically indistinguishable from its accumulated reaction products. The fact that small molecules can distribute rapidly between medium and suspended cells permits some simplifying assumptions. The agent in medium A, was considered to equilibrate between the cell cytosol and medium before a significant fraction of A, was consumed in medium, k14, k41 >> klz, k4. The concentrations [A,] and [&]at time zero were considered to be equal to the initial concentration of drug in medium, C,,. [A,], = Coe-(k12+keVJt

(2)

klZk26Vc + k45Vc(k25Vc + h 3 - k45Vc - k l Z ) e - ( k 1 2 + k e ~ ~ t + k&Vc)((Y- k45Vc - kiz)(P - k i z - k45Vc) kizk25Vc + k4sVA-u + biz + kz3) e-at + -a(kiz + k45VC - a)(P - a ) kizkz5Vc + k45Vc(-a + kiz + k d e-.] (3) -P(kiz + k45VC - P)(. - P) +

(Y and @ are the positive and negative solutions, respectively, to the quadratic equation ax2 + bx + c = 0, in which a = 1,b = kxVc + k , + k52 + 1223, and c = kz,V,k, + k2& + k23kW Equations 2 and 3 are the kinetic laws for this model. [Pel is the cumulative concentration of reaction product that is formed in the cell volume between initiation of drug exposure, to,and completion, t. The cell volume per milliliter of medium, V,, determines the relative contribution of cellular reactions to the total reaction rate and is calculated from the number of cells per milliliter and the cell diameter. The [Pel term for the intracellular methyldiazonium ion reaction products was calculated from measured rate constants, cell number, and initial agent concentration and related to cellular effect, in this case, growth rate inhibition. The lengthy eq 3 can be reduced to an approximate solution shown in eq 4 as several terms do not contribute significantly to

[P6lt =

the value of [P,]. Equation 4 was applied to substances that are converted to unstable metabolites, that is, where M2and Ms do not reach equilibrium between the cell and medium before reaction to product. Equation 5 can be applied to cell culture metabolism

I systems in which agents and metabolites are present in equal concentrations in the medium and cell volume and V, is small.

0

'

2

3

4

5

6

7

8

c e l l Concentration ( ~ 1 0c6e l l s / r r l )

Figure 2. DMN-OAc disappearancerate constants measured in cell culture medium and medium containing (1-8) X le cells/mL are plotted against cell concentration. The rate constant for the hydrolysis of DMN-OAc in medium is obtained from the y intercept, and the intracellular rate constant can be determined from the slope for any cell concentration. Under these assumptions, [P3It = [Pelt.

Results The activity of three metabolically activated agents were determined as the fractional inhibition of P388 growth rate relative to an untreated control cell population. Cells in suspension in serum-free medium, lo6 cells/mL, were treated with drug at 37 "C, p H 7.4. Two assay procedures were used in which cells were exposed to a drug concentration over a range of exposure periods or for a fixed time period with a range of drug concentrations. Growth rate inhibition data was compared to initial drug concentration, Cot incubation time, t, or [P6] for each experiment. Rate constants for disappearance of parent drug in medium and in medium containing a range of cell populations, (1-10) X lo6 cells/mL, were determined and plotted against cell numbers to calculate rate constants kI2 and k45 for extra- and intracellular metabolism, respectively. These data are shown in Figure 2 for DMN-OAc, where k12 = 0.00043 min-' and k4,V, = 0.0034 min-' a t lo6 cells/mL. The extracellular metabolism and/or chemical hydrolysis rate constant was increased through the addition of 300 units of AcChE to the medium. DMN-OAc was hydrolyzed by AcChE with a V,, of 0.0073 pM/min and K , of 25.8 pM. With the addition of 300 units of enzyme to medium, k12increased to 0.0156 min-I as shown in Table I. The intracellular metabolism rate constant was decreased by addition of the irreversible cholinesterase inhibitor paraoxon (13). At 1mM concentration, paraoxon treatment decreased growth rate of P388 cells by less than 10% but reduced k4Vc of DMN-OAc from 0.0034 to O.OOO6 min-' (Table I). These manipulations provided a means to alter cellular metabolism and cytotoxic activity in a way that could be related to values of [P6] by using the described kinetic model. The fractional growth rate inhibition, F = 1 - (treated/control), observed following 30-min exposure of normal and paraoxon-treated P388 cells to (100 pM) DMN-OAc is shown in Figure 3. The EDso(C) for DMN-OAc against normal P388 cells under these conditions was 12 p M , which was decreased substantially by inhibition of intracellular hydrolysis with paraoxon, EDN(C) = 80 f 29 pM.

Metabolically Activated Methylating Agents

Chem. Res. Toxicol., Vol. 2, No. 5, 1989 291

Table I. Rate Constants ( m i d ) for Cytotoxic Methylating Agents under Cell Culture Incubation Conditions DMN-OAc MNUT PBS PBS

culture medium kl2

+AcChE (300 units) k,Vc

+paraoxon (1mM) k23 k66 k62

k%VC

0.00043 0.0156 0.0034" 0.0006 1.7b 1.7b 41 0.014*

0.0026 0.0080 0.0030 0.0028 0.47c 0.47c 41 0.014

MAM-Ac PBS Fisher's 0.0002 0.010 0.018 0.0005 0.0005 O.OOld O.OOld 41 41 0.014 0.014

" V , for lo6 P388 cells/mL is 0.000345. *From ref 12. CCalculated from Fob. and eq 4 and 6. dFrom ref 7.

Time (min)

Figure 4. In the presence of 300 units of acetylcholinesterase, the effect of 30 pM DMN-OAc on the growth rate inhibition of is greater than the effect of 53 pM DMNnormal P388 cells (0) OAc on paraoxon-pretreated P388 cells (0). Each point shows the average and standard deviation of four measured values of F plotted against the exposure period.

'I

.9

OF 0

.

.

20

40

60

80

100

1

120

Initial Concentration (uM)

Figure 3. The growth rate inhibition,F, produced by DMN-OAc during a 30-min incubation period is greater for normal P388 cells (0) than for cells pretreated with paraoxon (0). Each point shows the average and standard deviation of four measured values of F plotted against the initial DMN-OAc concentration, C,,. Growth rate inhibition data for normal P388 and paraoxon-treated cells in AcChE-containing medium are shown in Figure 4. The protocol for this experiment varied exposure time between 2 and 60 min for 30 or 52 pM concentrations of DMN-OAc. In spite of the similarities in these experiments, only one data point of Figure 4 can be compared directly to Figure 3. At Co = 30 pM and t = 30 min, DMN-OAc in AcChE-containing medium had the same activity, F = 0.80 f 0.08, as in PBS, F = 0.78 f 0.08. This is a limiting problem in the use of cell culture assays for investigations of drug effects that changes over time of exposure. The use of an appropriate dose function, such as [PI, relates the time and concentration parameters so that all data points from these experiments can be compared. The MNUT (90 mM) reaction rate constant in 150 pM PBS, 37 "C,pH 7.4, was 0.0026 min-' measured by HPLC. Acetylcholinesterase hydrolysis of MNUT occurred with a V,, = 0.0017 pM/min and K, = 18.3 pM. Under cell incubation conditions, 300 units of AcChE increased k12 slightly t o 0.008 min-'. Intracellular rate constants were determined by incubating 90 pM MNUT in PBS with (1-8) X lo6P388 cells/mL. The rate constant k6Vc a t 106 cells/mL was 0.0030 min-l. Paraoxon (1 mM) pretreatment did not significantly change k6VV It is possible that intracellular hydrolysis of MNUT is a thiol-catalyzed

I

Time (min)

Figure 5. The addition of 300 units of acetylcholinesterase to PBS medium (0) did not change the growth rate inhibition of 68 pM MNUT against P388 cells in PBS medium (0).Each point shows the average and standard deviation of four measured values of F plotted against the exposure period. chemical reaction rather than an enzyme-mediated event (14). The effect of MNUT on P388 cell growth rate is shown in Figure 5. At an initial concentration of 68 pM, a 50% inhibition in the growth rate was observed after 130-min incubation in PBS. The same activity, EDm at 119 min, was observed in medium containing 300 units of AcChE. In contrast to DMN-OAc and MNUT, the metabolic intermediate generated from hydrolysis of MAM-Ac is reported to have substantial chemical stability (7). At neutral pH, 37 "C, MAM has a half-life of 12 h, kS = 0.0002 min-'. Hydrolysis of MAM-Ac by AcChE occurred readily with a V,, 0.027 pM/min and K, = 6.5 pM. This compound was also hydrolyzed rapidly in Fisher's medium

292 Chem. Res. Toxicol., Vol. 2, No. 5, 1989

Weinkam and Plakunou

a 7

6 F

5 4

.2 '31

/

3 2

, 1 1

00

50

100

150

200

250

300

350

400

450

500

Initial Concentration (uM)

Figure 6. The relation between P388 cell growth rate inhibition, F,and initial concentration is shown for MAM-Ac after 120- (0) and 2880- (0) min incubation in PBS and Fisher's medium, respectively.

containing 10% donor horse serum. A plot of the observed rate constant in PBS medium containing (2-10) X 106 P388 cells/mL had a shallow slope, however, with kaVc = 0.0005 m i d a t 106 cells/mL. Rate constants in PBS and Fisher's medium are shown in Table I. Growth rate inhibition activity is shown in Figure 6 for two different experiments. Activity was plotted against initial concentration of drug in PBS for a 2-h incubation period and in Fisher's medium for a 48-h period. It was necessary to use Fisher's medium containing 10% donor horse serum to maintain cell viability over the 48-h period. Neither added AcChE or paraoxon pretreatment caused a significant change in the curve for the 48-h experiment.

Discussion The objective of this paper is the demonstration of the utility of the kinetic model to compute a function that can be reasonably related to observed inhibition of cell growth rate. For the methylating agents being investigated, this function was [Pelt, the accumulated intracellular reaction products. All of the parameters necessary for calculating [Pelt for DMN-OAc are known or were estimated. The stability of the metabolite N-methyl-N-(hydroxymethy1)nitrosamine (DMN-OH) has been determined by using a flow-trapping method (12).The rate constant for alkylation at pH 7.4, 37 "C, is 1.7 min-', and the half-time is 21 s. The half-time for partitioning of the small, rather lipophilic molecules, DMN-OAc, DMN-OH, and others in Table I, was estimated to be around 1 s (1.516). The rate constants k25 = k52 = 41 s-l were used in all calculations. Substitution of the rate constants of Table I and experimental parameters Cot t , and V, into eq 4 gave values for [Pel for each experimental point shown in Figures 3 and 4. These were plotted against growth rate inhibition in Figure 7. The curves for each experiment were the same within experimental error. Furthermore, the range of activities, EDm(P)= 135-160 pM, was the same as that observed for MNU, streptozotocin, and MNNG (31, which also act through the methyldiazonium ion intermediate. The convergence of lines in Figure 7 supported the accuracy of the kinetic model and rate constant parameters, as did the similarity in activity between DMN-OAc and

0 0

50

100 150 200 Concentration (uM)

250

0

Figure 7. When the growth rate inhibition, F,of DMN-OAc is related to [Pel,no significant difference in activity is seen against normal P388 cells in medium (0), in medium containing 300 units of acetylcholinesterase (a), for paraoxon-treated P388 cells in medium (A),or for paraoxon-pretreated cell in medium containing 300 units of acetylcholinesterase (v). previously studied compounds. This represents another example of growth rate inhibition by activity of methyldiazonium ion alkylation which is the same in spite of its formation from different precursors. The similarity between previously reported DMN-OAc and the methylnitrosourea data also permitted use of the following empirically derived relation between growth rate inhibition, F , and [PI to predict cellular activity under different experimental conditions (3):

F = (4 x 10-3)[P6]- (4.6 X 104)[P6]2 ([Pel in pM) (6) I t was observed experimentally that inhibition of intracellular hydrolysis by paraoxon pretreatment substantially decreased DMN-OAc activity while increasing extracellular metabolism by added AcChE had no effect. These facts were consistent with the kinetic model even though the stability of the metabolite DMN-OH, k , = kM = 1.7 min-', is great enough to permit a large fraction of intracellular material, M5, to partition from the cell volume to medium before reaction to form intracellular methyldiazonium ion. Discussion of this process can be simplified by reference to calculated and observed values for DMN-OAc at a single set of conditions: initial concentration of 30 pM, 30-min incubation period, and a cell density of 106 cells/mL (Table 11). Under normal P388 cell conditions only 13% of DMN-OAc was hydrolyzed in 30 min, but 89% (3.5 nmol), of this occurred in the cell volume. The calculated values [Pel30 = 338 pM, Fdc = 0.82, were consistent with the observed activity, Fobs = 0.80. The value of (Pel30 = 0.12 nmol was much smaller than the 3.5 nmol formed within the cell, indicating that greater than 95% of MS was exported from the cell volume. Virtually no Mz was imported, as estimated by calculating [Pel30 = 0.38 pM with kd5 = 0, due to the small fractional cell volume V , = 0.00034. Since the bulk of intracellular product resulted from intracellular activation, it is clear that inhibition of cellular hydrolysis by paraoxon pretreatment will strongly effect activity (Table 11, column 3). Addition of AcChE substantially increased the fraction of DMN-OAc hydrolyzed from 13 to 44% by increasing extracellular hydrol-

Chem. Res. Toxicol., Vol. 2, No. 5, 1989 293

Metabolically Activated Methylating Agents Table 11. Cell Culture ParametersCalculated for DMN-OAc Activity against P388 Cell Growth Rate When Incubated at Co= 30 pM and t = 30 min

klz, min-' k16Cc, mi& k,Vc/(kl, + k46VC)

([All, [All,)/ [A110 [ P B I ~PM , (P6)30rnmol Fdc

P388 0.00043 0.0034 0.89

+AcChE/ paraoxon +paraoxon +AcChE (300 units/ (1 mM) (300 units) 1 wM) 0.0156 0.00043 0.0156 0.0034 0.0006 0.0006 0.18 0.04 0.58

0.13

0.03

0.44

0.37

338 0.12 0.82 0.80 f 0.13

60 0.021 0.22 0.30 f 0.10

27 1 0.093 0.74 0.80 f 0.11

48 0.016 0.18 0.22 0.09

Fob ysis. Since only around 0.1% of extracellular M2 gained access to the intracellular volume, this had little impact on calculated [P6]30 = 271 pM and Fdc = 0.74 and was consistent with the lack of experimentally observed difference in growth rate inhibitors, Fob = 0.80. Only when intracellular hydrolysis was inhibited by paraoxon did the AcChE-derived product contribute a sizable fraction of the low P6 concentration (Table 11, column 5). It is clear from these results that a cell culture activation system that is exclusively extracellular, such as the S-9 activation of dimethylnitrosamine, would appear to be much less active than DMN-OAc, even though they have a common intermediate, DMN-OH, and the intrinsic activity of the ultimate active methyldiazonium ion may be the same. This is related to the small fractional cell volume present in a cell culture experiment using a few million cells per milliliter of medium. Cell culture assay conditions in which cells constitute a very small fraction of the total volume are almost the reverse of the in vivo or whole tissue environment in which cells are contiguous with little interstitial volume. In this case, export of an intracellularly formed intermediate, such as DMN-OH, would represent cell-to-cell transfer with limited loss to interstitial fluids or capillary blood flow prior to formation of the methyldiazonium ion. Methylnitrosourethane is activated by hydrolysis of the ester bond to give a metabolite, N-methyl-N-nitrosocarbamic acid (Figure 1). The stability of this intermediate is not known. Decarboxylation of N,N-diarylcarbamic acids occurred with rate constants in the range of 1min-' a t neutral pH (17). This value could be used for k23 and k56. Conversely, the relationship between F and [P6]t expressed in eq 6 can be combined with eq 4 to calculate .,k When kb2 = 41 min-' was used, the calculated value ks = 0.46 min-' was close to the published values for N,N-disubstituted carbamic acids. The calculated stability of N-methyl-N-nitrosocarbamic acid, t l = 1.5 min, was 3 times longer than that of DMN-dH. While this reduces the efficiency with which intracellular activation causes cell damage, this remains the major pathway for accumulation of reaction product. The insensitivity of this compound to AcChE-enhanced extracellular activation was similar to that of DMN-OAc. The metabolic activation of MAM-Ac proceeds through formation of MAM. This intermediate has a significant chemical stability, tl12 = 1 2 h, 12% = 0.001 min-'. In contrast to DMN-OH and N-methyl-N-nitrosocarbamicacid, MAM should have sufficient stability to equilibrate between cell volume and medium. The data of Figure 7 show growth rate inhibition after a 48-h incubation period. The EDM(C)was 175 p M . Under these conditions 95% of MAM-Ac has been hydrolyzed in the 48-h period so that

EDw(C) was essentially equal to EDw(P). The extracellular hydrolysis rate k12 = 0.001 min-' was 20 times that of k&Vc so that 95% of MAM was formed in the medium. Neither paraoxon inhibition of intracellular hydrolysis nor enhanced AcChE metabolism altered the observed effect. The growth rate inhibition data were closely modeled by eq 6 with [PI equal to Co (Figure 7). In the 2-h exposure to MAM-Ac in PBS medium, very little activity was observed as the extracellular hydrolysis was very slow, k12 = 0.0002 min-', and only 8% of MAM-Ac was activated. Under these conditions, intracellular activation was the most important pathway, k6/(k6Vc + k12) = 0.71. It may be seen from Figure 7 that the predicted growth rate inhibitor was 2 times less than that which was observed. MAM may be subject to further intracellular metabolic activation through oxidation of the carbinol. This process may increase ks to approximately 2 times the chemical hydrolysis rate and account for the high observed activity of MAM-Ac (18, 19). The kinetic model that was developed to relate the cell culture assay of growth rate inhibitor of metabolically activated agents has been demonstrated to provide a reasonable model of experimentally observed biological effects. Use of the models can provide new insight into the process of metabolic activation involving the distribution of intermediates between medium and cell volumes by enabling the quantitative importance of these parameters to be estimated. This approach has apparent value in the interpretation of cell culture assay data and in the application of these results to the living animal.

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).

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