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TOXICOLOGY AND APPLIED PHARMACOLOGY 97,480-488 (1989)

Genotoxicity of N-Nitrosochlordiazepoxide in Cultured Mammalian Cells’

GIOVANNIBRAMBILLA,*"LUIGIROBBIANO,*ANTONIETTAMARTELLI,*ENRICACAJELLI,* ALESSANDRAALLAVENA.*ANDMAUROMAZZEI**

*Institutes ofPharmacology and OJ **Pharmaceutical Sciences, University ofGenoa, I- 16132 Genoa. Italy

Received May I-7, 1988; accepted October 3. 1988

Genotoxicity of iliNitrosochlordiazepoxide in Cultured Mammalian Cells. BRAMBILLA. G..

ROBBIANO, L., MARTELLI. A., CAJELLI, E., ALLAVENA, A., AND MAZZEI. M. (I 989). Toxicol. Appi. Pharmacol. 97,480-488. Chlordiazepoxide. a benzodiazepine derivative commonly used

for the treatment of anxiety. was found to react with sodium nitrite in HCI aqueous solution yielding, at pH ranging from 0.5 to 5. N-nitrosochlordiazepoxide (NO-CDE). In the absence of

a metabolic activation system. a dose-dependent frequency of DNA single-strand breaks was revealed by the alkaline elution technique in V79 cellsexposed to subtoxic NO-CDE concentrations ranging from 33 to 330 PM. DNA lesions were only partially repaired within 48 hr. and

their promutagenic character was demonstrated by the induction of 6-thioguanine resistance in the same cells. The genotoxicity of NO-CDE was confirmed by results obtained in metabolically

competent primary cultures of both rat and human hepatocytes. which displayed similar dose- related amounts of DNA fragmentation and of DNA repair synthesis after treatment with concentrations ranging from 33 to 1000 PM. In conditions similar to those which might occur in

the stomach of a patient taking chlordiazepoxide the concentration of NO-CDE in the reaction mixture (50 FM) was of the same order as the concentrations found to induce a genotoxic effect in cultured mammalian cells. 2’ 1989 Academic press. IIIC

The possibility that carcinogenic N-nitroso compounds are formed in vim from the reaction of drugs containing amino groups with nitrite present in the gastric environment has been demonstrated in a large number of ani- mal experiments (Mirvish, 1975: Lijinsky and Taylor. 1977; IARC, 1980a,b). Such interaction should be favored by the acidity of human gastric juice. A series of data recently summarized by Bartsch and Montesano (1984) unequivocally gives proof that N-ni-

’ This work was supported by Consiglio Nazionale delle Ricerche Special Projects “Oncology” (Contract 86.00326.44), “Preventive and Rehabilitative Medicine”

(Contract 86.01877.56), and by funds of the Public In- struction Ministry (Italy).

’ To whom correspondence should be addressed at: Is-

tituto di Farmacologia dell’Universiti, Viale Benedetto XV. 2, I- 16 I32-Cienova, Italia.

troso compounds are generated in the human body, even after ingestion of levels of precursors considered to be normal, and provides evidence that exposure to nitrosamines will lead to an increased probability of cancer in humans, Consequently, the need of verifying the risk to human health arising from the intake of nitrosatable drugs has become evident in recent years (IARC, 1980a: Brambilla, 1985).

The following experimental findings prompted us to further investigate the formation and the genotoxicity of the nitrosation products of chlordiazepoxide, a benzodiazepine derivative used for the treatment of anxiety that is widely prescribed (Blackwell, 1973). Walser cf al. (1974) first reported that treatment of chlordiazepoxide with sodium nitrite yields in acetic acid a nitrosoamidine, i.e.. 2-N-nitrosochlordiazepoxide (NO-CDE),

0041-008X/89 $3.00 Copyright 8 1989 by Academic Press, Inc. All rights of reproduction in any form resewed.

480

N-NITROSOCHLORDIAZEPOXIDE GENOTOXICITY IN MAMMALIAN CELLS 481

and in hydrochloric acid a dihydroquinazo- line. Induction by NO-CDE of mitotic gene conversion in S. cerevisiae was observed in a host-mediated assay by Krafft and Onken (1976). Preda et al. (1976) demonstrated the presence of the N-nitroso derivative in the stomach and in the small bowel of rats fed with chlordiazepoxide and nitrite, and Lijin- sky and Taylor ( 1977) detected in similar experimental conditions a marginally signifi- cant increase in the incidence of nervous system tumors. Induction of mutation was observed by Andrews et al. (1980) in five S. typhimurium strains after exposure to the chlordiazepoxide nitrosation mixture. The mutagenic effect was more evident by the absence rather than by the presence of a metabolic activation system, as subsequently reported by Takeda and Kanaya (198 1). Fi- nally, DNA fragmentation was detected (Brambilla et al., 1985) in CHO cells after exposure to an -0.2 mh4 concentration of chlordiazepoxide nitrosation products.

Recently Mazzei et al. (unpublished data) found that the reaction of chlordiazepoxide with nitrite yields NO-CDE, not only when performed in acetic acid as reported by Walser et al. ( 1974) but also when carried out in aqueous HCl solution. The measurement by HPLC analysis of NO-CDE formation at pH ranging from 0.5 to 5.0 revealed that the yield reaches its maximum at pH 3.5. This yield was approx 55% of the theoretical one when 40 mg of chlordiazepoxide hydrochloride was reacted with 20 mg of NaNOz in 10 ml of HCl (pH 3.5) at 37°C for 1 hr in the dark.

The present work was undertaken to evaluate the genotoxicity of NO-CDE in some short-term in vitro assays on mammalian cells.

METHODS

Chemicals. NO-CDE was synthesized by Mazzei as described by Walser et al. (1974). In brief, over a period of 30 min NaNOz (0.26 mol) was added in small portions to a stirred solution of chlordiazepoxide (0.20 mol) in

400 ml of glacial acetic acid, and the reaction mixture was left at room temperature with stirring for another 30 min. The precipitate was recrystallized from ether/ hexane. NO-CDE (98-99% pure: 75% yield) was the only product obtained (melting point 158-160°C dec.). Kept at -20°C as a solid, NO-CDE was found to be chemically stable for 6 months. In aqueous solution, under conditions corresponding to those of the experiments carried out to evaluate its biological activity, NO-CDE was found to be stable for 24 hr, as revealed by HPLC analysis. N-Nitrosodimethylamine (DMN) and tetraethylam- monium hydroxide were purchased from E. Merck (Darmstadt, FRG): N-nitroso-N-methylurea (NMU) from Serva Feinbiochemica (Heidelberg. FRG); N-lauro- ylsarcosine sodium salt (Sarkosyl), 6-thioguanine. and collagenase type I from Sigma Chemical Co. (St. Louis. MO): a-modified minimum essential medium (a- MEM). Williams’ medium E (WME) and fetal calf serum from Flow Laboratories, Milan, Italy; [meth$3H]thymidine (sp act 23-25 Ci/mmol) from the Radiochemical Centre (Amersham, UK). All other chemicals, reagent grade, were obtained from E. Merck (Darmstadt, FRG).

Cell culture and treatment. V79 cells (NIH. Coriell In- stitute for Medical Research, Camden. NJ), an estab- lished line from Chinese hamster lung, were cultured in a-MEM supplemented with 5% fetal calf serum. Di- methyl sulfoxide was used as a solvent for NO-CDE and was added at the same concentration in control cultures.

For the determination of cytotoxicity. 6 X lo5 cells were inoculated in 25cm’ plastic flaks and after 24 hr were exposed to the test compound for 60 min in serum- free medium. Cell viability was measured immediately after exposure either by trypan blue dye exclusion or by cloning efficiency. In the trypan blue assay, at least 1000 cells/sample were examined. In the colony formation assay, cells were trypsinized at the end of treatment and inoculated in plastic dishes (200 cells/dish); after incubation for 7 days, colonies were fixed, stained. and counted.

For the assay of DNA-damaging activity, 24 hr after seeding V79 cells were labeled with 0.1 &i/ml of [methyl-3H]thymidine for 24 hr. Cells were then exposed to the test compound for I hr in nonradioactive serum-free medium and assayed for DNA fragmentation either immediately or after incubation in complete medium at 37°C for 24 and 48 hr.

Rat hepatocytes were isolated from Sprague-Dawley male albino rats (200-250 g) by collagenase perfusion, as described by Williams (1977). Human hepatocyte suspensions were prepared from fragments of human liver discarded during the course of prescribed surgery essentially according to Strom el al. (1982). Case 1 was a 60- year-old male who underwent surgery for hepatic metas- tases of a colon carcinoma. Case 2 was a 57-year-old male who underwent surgery for an echinococcus hydatid cyst. The proportion of viable cells after perfusion, as measured by trypan blue exclusion, was 77% in Case 1 and 75% in Case 2.

482 BRAMBILLA ET AL.

Isolated rat or human hepatocytes were suspended in

WME supplemented with 10% fetal calf serum and gen- tamycin (50 pg/ml), and aliquots of these suspensions were plated in plastic dishes as follows: 2 X IO6 cells in

60-mm uncoated dishes for DNA fragmentation assay,

and I X IO6 cells in 35-mm dishes coated with rat tail collagen for determination of cell viability and the un-

scheduled DNA synthesis (UDS). After attachment for 3 hr. cells were incubated for 20 hr with the test compound in serum-free medium. DMN was used as the positive

control to verify the capability of hepatocytes to activate

procarcinogens. DNA fragmentation and UDS were measured immediately after exposure to the test com-

pound. The choice for the hepatocytes of a 24-hr exposure, instead of 1 hr as for V79 cells, was dictated by the

opportunity to adhere to the routinely employed proto- col developed by Williams et a/. (1982) for the hepatocyte

primary culture/DNA repair assay. and thus allow a meaningful comparison with the amounts of unscheduled DNA synthesis elicited by other N-nitroso com-

pounds. It is obvious that this choice. while making it

possible to obtain some information on the influence of the length of treatment, at the same time. makes a com-

parison of the results provided by the two cell types difficult.

Determirlation qf DNkdamaging activity. In both

V79 cells and rat or human hepatocytes the presence of DNA fragmentation (single-strand breaks. and/or alkali-

labile sites) was evaluated by the alkaline elution technique (Kohn et al., 1976). as previously reported (Bram-

billa e/ al.. 1978). The cell suspension (- IO6 cells) was deposited onto a Millipore filter (mixed esters of cellu-

lose: 25-mm diameter, 5-hrn pore size), washed with cold Merchant’s solution (NaCI 0.14 M, KC1 2.7 mM.

KHZP04 1.47 mM, Na2HP0, 8.1 mM, NazEDTA 0.53 mM; pH 7.6). and lysed at room temperature (20-22°C)

with 0.2% sodium lauroylsarcosinate (Sarkosyl). 2 M NaCl, and 20 mM NazEDTA (pH 10). Single-stranded

DNA was eluted from the filter in the dark, at a pump speed ofO.13 ml/mitt. with 13 ml of60 mM tetraethylam-

monium hydroxyde and 20 mM NazEDTA (pH 12.3). Eluted fractions werecollectedat IO-min intervals. In the

experiments performed with V79 cells. the DNA content ofthe IO eluted fractions and that remaining on the filter

was dosed radiometrically by liquid scintillation counting. In the experiments performed with rat or human he-

patocytes the DNA content was determined by a previously described microfluorimetric procedure (Bram- billa ef al.. 1978). Data are expressed as the percentage of

DNA eluted from the filter which gives direct information about the experimental results obtained. The aver-

age elution rate over controls (k; ~ &) is also reported: K, is the elution rate of DNA from treated cells and & the elution rate from corresponding controls. K values

were calculated from the equation K = (-In FR)/T: where FR is the fraction of DNA retained on the filter and I’is the eluting volume (I 3 ml). As a first approxima-

tion K is directly proportional to the frequency of DNA

breaks (Kohn et al., 1976). DNA repair te,st. The procedure used to evaluate the

induction of DNA repair synthesis was essentially that

described by Williams (1977). Rat or human hepatocyte primary cultures were simultaneously exposed to NO-

CDE and [meth$3H]thymidine (10 &i/ml). After 20 hr incubation the cultures were washed twice with 0.9% NaCl and treated with 1% sodium citrate solution for 10

min. The cells were fixed in acetic acid:ethanol (1:3) for three IO-min changes. A section of the air-dried culture

dish bottom was cut out, glued to a microscope slide. dipped in Kodak NTB-2 emulsion, and exposed for 7 days at 4°C. Autoradiographs were developed and stained with May-Gruenwald-Giemsa. Autoradio-

graphic nuclear grains were counted in 50 consecutive cells of each slide. The highest cytoplasmic background

count for each cell was subtracted from the nuclear count to obtain the net nuclear grains due to DNA repair syn-

thesis. The data are expressed as the means of the 100 net nuclear counts obtained from two autoradiographs f SD. When the increase in the average number of net

nuclear grains exceeded five as compared with the corresponding control. DNA repair induction was estimated

to be positive. Determination c?f’mutagenic artivii~: The quantitative

assay for induced frequency of 6-thioguanine-resistant (TG’) cells was a modification (Cajelli et al.. 1987) ofthat described by Myhr and Di Paolo (1978). The duration of

exposure to the test chemical was 1 hr. At the end of the h-day period of mutant expression, the fraction of TG’ cells in the population was determined from IO dishes

( 100 mm) by counting mutant colonies 6-7 days after seeding 3.5 X IO5 cells per dish. For selection of mutants,

h-thioguanine was added 2 hr after seeding to give a final concentration of 10 pg/ml.

RESULTS

Because of the low solubility, the maximal concentration of NO-CDE which could be tested was 1 mM. Data of Table 1 show that a dose-dependent reduction of V79 cells plating efficiency was produced by 1 -hr exposure to concentrations ranging from 10 to 330 PM. In contrast, when measured by trypan blue exclusion, cell survival was practically unaffected at the same concentrations and only minimally decreased at 1 mM. Taking into account that with plating efficiency what is measured is the effect on the capacity of cell

N-NITROSOCHLORDIAZEPOXIDE GENOTOXICITY IN MAMMALIAN CELLS 483

TABLE 1

RELATIVE SURVIVAL OF V79 CELLS AND OF RAT OR HUMAN HEPATOCYTES AFTER EXPOSURE TO NO-CDE

NO-CDE (N)

Relative survival of V79 cells

Trypan blue Plating exclusion efficiency

Relative survival of hepatocytes (trypan blue exclusion)

Rat Man

0 (0.99 f 0.01) (0.82 * 0.04) (0.96 k 0.02)” (0.98-0.96)h 10 0.99 -e 0.0 1 0.81 F 0.09 0.98 f 0.01 33 0.99 + 0.0 1 0.68 -+ 0.07 0.98 k 0.03

100 0.98 f 0.01 0.42t0.11 0.94 k 0.05 0.99-0.95 330 0.97 Ik 0.01 0.33 + 0.08 0.92 +- 0.06 0.99-0.86

1000 0.88 + 0.02 0.84 zk 0.11 0.95-0.85

’ Data are the means f SD of at least three independent assays. Values in parenthesis indicate the fraction of viable cells in controls. Relative survival was calculated from the ratio (fraction of viable cells in treated cultures/fraction of viable cells in control cultures).

’ Data obtained from two human donors (Case 1 -Case 2).

for unlimited growth, while trypan blue ex- DNA-damaging potency of NMU, a known clusion is an indicator of cell membrane in- carcinogen used as positive control, was of tegrity (Roper and Drewinko, 1976) it is not the same order of that of NO-CDE. To deter- surprising that doses greater than those which mine whether DNA damage was repaired, suffice to damage reproductive integrity were V79 cells treated with 100 PM NO-CDE were needed to produce immediate cell death, as incubated at 37°C for 24 and 48 hr after NO- revealed by trypan blue inclusion. In primary CDE removal, and the amount of DNA frag- cultures of both rat and human hepatocytes mentation was then measured. The results a 20-hr exposure to NO-CDE determined a obtained are shown in Table 2; they indicate limited and variable reduction in the fraction that DNA lesions were only partially re- of trypan blue-excluding cells only at concen- moved after 48 hr of posttreatment incuba- trations ranging from 100 to 1000 PM. tion.

DNA-Damaging Activity and Induction Qf DNA Repair

Data provided by DNA damage/alkaline elution assay performed on V79 cells are listed in Table 2. After a 1 -hr exposure to NO- CDE the amount of DNA fragmentation was dose dependent for concentrations ranging from 33 to 330 PM. The occurrence of DNA damage at doses not reducing the fraction of viable cells, as evaluated by trypan blue exclusion, indicates that DNA lesions are the specific consequence of NO-CDE-DNA in- teractions and not an aspecific event due to a cell death associated endonucleolytic DNA degradation (Williams ef al., 1974). The

Data provided by the DNA damage/alkaline elution assay, performed to evaluate the DNA-damaging activity of the chlordiazepoxide nitrosation product in primary cultures of rat and human hepatocytes. are shown in Table 3. When rat hepatocytes were treated for 20 hr. concentrations of NO-CDE ranging from 33 to 1000 PM were found to produce a dose-dependent increase of DNA elution rate, i.e., of DNA fragmentation. In identical treatment conditions NO-CDE produced a similar dose-dependent amount of DNA fragmentation in primary cultures of human hepatocytes obtained from donor No. 2: un- fortunately the number of liver cells obtained from donor No. 1 was insufficient for this test.


TABLE 2

DNA FRAGMENTATION IN V79 CELLS AFTER I-hr EXPOSURE TO NO-CDE

Treatment conditions

No. of samples

% DNA eluted

from the filter (mean + SD)

El&on rate

over controls

(Kt - K-c)

Significance

level”

(P)

Control 10 9.6 k I .6 (A; = 0.0078) NO-CDE 33 pM 6 17.6 k 1.8 0.007 1 to.002 NO-CDE 100 PM 6 38.02 3.1 0.0290 <0.002

6 23.1 f 2.2" 0.0124 -co.002 6 19.3 k 2.7' 0.0087 <0.002

NO-CDE 330 PM 6 78.1 + 5.2 0.1091 -co.002 NMU 330 PM 5 67.3 ?I 4.9 0.0782 <0.002

a Significance level was determined by the use of the nonparametric Wilcoxon two-sample two-tailed test (Riimke and De Jonge, 1974).

‘.’ Values obtained after a posttreatment incubation interval of 24 hr (h) and 48 hr (c‘).

Table 4 lists the results obtained by the unscheduled DNA synthesis. It is evident that evaluation of DNA repair in rat and human substantial differences in the mean grain hepatocyte primary cultures exposed to NO- counts were observed both in the three inde- CDE for 20 hr. In both species concentrations pendent experiments carried out with rat he- ranging from 33 to 1000 PM produced a sim- patocytes and in the assay performed with ilar dose-related increase in the amount of liver cells from two human donors. However,

TABLE 3

DNA FRAGMENTATION IN RAT AND HUMAN HEPAT~CYTE PRIMARY CULTURES

AFTER IO-hr EXPOSURE TO NO-CDE

Treatment conditions

No. of samples

‘3% DNA eluted

from the filter (mean *SD)

Elution rate

over controls

K - Kc)

Significance level” ( p)

Control NO-CDE 33 yM

NO-CDE 100 FM NO-CDE 330 PM

NO-CDE 1000 PM DMN 5 mM

Control

NO-CDE 100 PM NO-CDE 330 pM NO-CDE 1000 PM DMN 5 mM

Rat hepatocytes

26.6 + 2.3 (Kc ~0.0238) 30.9 i 3.9 0.0046

38.2 f 3.0 0.0132 56.7 A I 1.4 0.0406 71.8* 8.5 0.0736

76.0+ 8.1 0.0863

Human hepatocytes (Case 2)

32.9 i- 2.7 (Kc= 0.0307) 49.4 f 2.0 0.0217 61.0t 3.3 0.04 I7 62.8 f 3.7 0.0454 54.9 f 3.9 0.0306

-co.002 -co.002 <0.002 <0.002 -co.002

=0.1 =o. 1 =O.l =o. 1

a Significance level was determined by the use of the nonparametric Wilcoxon two-sample Iwo-tailed test (Riimke and De Jonge, 1964).

N-NITROSDCHLORDIAZEPOXIDE GENOTOXICITY IN MAMMALIAN CELLS 485

TABLE 4

DNA REPAIR SYNTHESIS IN RAT AND HUMAN HEPATWYTE PRIMARY CULTURES AFTER 20-hr EXPOSURETO NO-CDE

Treatment conditions No. of net nuclear grains (mean t SD)

Rat hepatocytes Expt I Control 2.2 f 2.9 (20) NO-CDE 33 /.LM 5.7 k 4.2* (56)

NO-CDE 100 /.LM 12.0 It 9.5* (75)

NO-CDE 330 +M 32.41 17.1*(96) NO-CDE 1000 #M 37.5 2 17.7* (100)

DMN 5 mM 31.5 2 l&1*(98)

Human hepatocytes Case 1 Control 1.1 + 3.8 (19) NO-CDE 33 fiM 3.5 t 3.7* (43) NO-CDE 100 /.LM 7.4 f 12.2* (69)

NO-CDE 330 HIM 10.5 ? 5.1* (90) NO-CDE 1000 fiM 31.0, 15.9*(97) DMN 5 mM 9.0 * 4.5* (87)

Expt 2 Expt 3 -0.4 + 3. I (6) 0.2 i 3.3 (8)

3.5 +- 3.5* (32) 4.1 f 4.2* (45) 3.1 r 2.9* (29) 7.0 2 5.6* (67)

4.2 k 3.0* (45) 13.3 + 7.2* (92) 14.6 _t 7.2* (97) 31.9* 14.1*(100) 8.5 F 5.7* (79) 18.0+ 11.2*(94)

Case 2 5.5 ?I 8.6 (42)

-

20.1 + 12.7* (92) 27.1 i 17.1* (90) 45.3 f 19.1* (94) 17.8 + 12.0* (97)

a Net nuclear grain counts of duplicate autoradiographs. Grain counts include cells with no nuclear labeling en- countered in the 50 cells counted for each slide. Numbers in parentheses are the percentage of cells with five or more nuclear grains.

* p f 0.001 vs controls; significance level was determined by the use ofthe Student I test (two-tailed).

these variations are in the order of those reported by others (Williams, 1977; Strom et al., 1983). The constant occurrence of DNA damage and repair in rat and human hepatocytes exposed to the procarcinogen DMN, used as a positive control, demonstrates their metabolic competence.

Mutagenic Activity

Data listed in Table 5 indicate that a I-hr exposure to NO-CDE in the absence of a metabolic system produced a dose-dependent increase in the number of TG’ clones. At the highest concentration tested (330 PM) the average mutation frequency was approximately 23 times higher than that in controls. NMU, used as positive control, yielded, at 1 mM concentration, a lower frequency of TG’ vari- ants.

DISCUSSION

The results of this work indicate that the reaction of chlordiazepoxide with nitrite, car-

ried out in HCl aqueous solution, at pH ranging from 0.5 to 5.0, produces NO-CDE. With a molar ratio drug/NaNO* of l/2.4, the maxi-

TABLE 5

INDUCTION 0F MUTATION pro 6-THIOGUANINE RESISTANCE IN V79 CELLS BY I-hr EXPOSURE TO NO- CDE

Treatment conditions TG’ mutants/ lo6 survivors”

Control 18.4 t 0.4 NO-CDE 10 ).LM 25.1 zk 2.6 (71-88Yio)”

NO-CDE 33 /.tM 30.0 t 4.5 (62-7610) NO-CDE 100 PM 105.8 + 51.6* (35-55s) NO-CDE 330 pM 415.6 + 84.0* (28-4310)

NMU 300 &LM 46.5 rt_ 5.3* (82-88%>) NMU 1000 LLM 130.3 + 33.3* (50-72s)

u Values are the means + SD of data obtained from three independent experiments.

’ Numbers in parenthesis indicate relative survival range.

* p < 0.01 vs controls; the significance level was determined according to Kastenbaum and Bowman ( 1970).


ma1 yield of NO-CDE (55% of the theoretical one) was obtained at pH 3.5.

NO-CDE concentrations ranging from 33 to 1000 pM were found to produce dose-dependent genotoxic effects in some short-term tests routinely employed to evaluate the potential carcinogenic activity of chemical agents. DNA single-strand breaks were observed in V79 cells; the repair of these lesions was largely incomplete after 4X hr, and their promutagenic character was demonstrated by the induction of mutation to TG’ in the same cells. Both DNA fragmentation and unscheduled DNA synthesis were detected in rat hepatocytes, and it is worth noting that NO- CDE exerted similar effects in primary cultures of liver cells obtained from two human donors. In contrast, any evidence of genotoxic effects (results not shown in the tables) was absent in parallel assays carried out with the parent compound chlordiazepoxide up to the maximal soluble concentration (1 mM).

Since a satisfactory correlation has been found to exist between the tumorigenic activity and the capabilities of inducing DNA fragmentation (Swenberg et al.. 1976: Sina et [II., 1983) DNA repair (Williams, 1977; Wil- liams et a/., 1982) and somatic mutations (Bartsch et al., 1980; Bradley ct a/., 198 1 ), our findings, which are in good agreement with the previously acquired evidence of NO-CDE genotoxicity (see Introduction), contribute to support the hypothesis that this N-nitroso compound may initiate a carcinogenic pro- cess. This hypothesis is strengthened by the comparison of the genotoxic potency of NO- CDE with those of the known carcinogens NMU and DMN. In fact, the potency of NO- CDE was of the same order of that of NMU in assays performed on V79 cells and greater than that of DMN in rat and human hepatocytes. Moreover. the DNA-damaging and mutagenic activities exerted by NO-CDE on V79 cells in the absence of a metabolic system indicate that it behaves as an activation- independent agent, and in turn this suggests that it may act directly as an ultimate carcino-

gen in the site of its production, i.e., at the level of gastric mucosa.

The usual therapeutic dosage of chlordiazepoxide is 25 mg/day, administered in di- vided doses without observing a temporal re- lation with meals. The concentration of nitrite in human gastric juice has been found to vary from 0 to 3.2 PM in fasting healthy volunteers (Raisfeld-Danse and Chen, 1983) and to depend on gastric pH in patients with stomach pathology, ranging from 1.7 to 4.6 pM at pH < 3 and from 30 to 300 pM at pH > 5 (Kyrtopoulos et al.. 1985). High nitrite concentrations are normally present in the achlorhydric stomach with the highest level reported being 600 yM (Tannenbaum et al.. 1979). After a meal the gastric concentration of nitrite seems to depend essentially on the salivary contribution. which in turn is related to the nitrate and nitrite concentration of the diet (Klein el a/., 1978). For U .S. residents the estimated amounts of nitrate and nitrite in- gested daily correspond to 99.8 and 1 1.2 mg, respectively (Tannenbaum et al., 1977). Wal- ters ( 1980) demonstrated that after consump- tion of meals containing 15.9 mg of nitrite the gastric juice nitrite concentration was raised to about 300 pM. The reaction of 25 mg of chlordiazepoxide with 2 mg of NaN02, carried out at 37°C in 40 ml of HCl solution (pH 3.5) yielded about 0.6 mg of NO-CDE (2.5% ofthe theoretical yield), which corresponds to an -50 PM concentration of this N-nitroso derivative in the reaction mixture. Therefore, in conditions very similar to the possible gastric environment of a patient taking chlordiazepoxide, NO-CDE is formed in a concentration of the order of those found to induce DNA damage and mutations in mammalian cells. In this respect, it is noteworthy that DMN. which is about five times less potent than NO-CDE in producing DNA damage and repair in both rat and human hepatocytes, was shown to induce rat liver cell carci- nomas in a dose-related frequency when administered chronically with the diet in dos- ages ranging from 0.2 to 5 mg/kg of body weight (Terracini et al.. 1967).

IV-NITROSOCHLORDIAZEPOXIDE GENOTOXICITY IN MAMMALIAN CELLS 487

Nitrosation reactions depend on the relative concentrations of substrate, catalysts, and inhibitors and are influenced by the in- homogeneity of the stomach contents, by pH and by other factors. Thus our data do not allow any prediction of NO-CDE yields in patients ingesting chlordiazepoxide. However, they suggest that at least in some individuals NO-CDE might be formed in amounts sus- ceptible of inducing genotoxic effects. This might occur, for example, in patients treated with gastric antacids and/or H,-blocking agents which should enhance chlordiazepoxide nitrosation either directly by raising the pH of gastric environment to values favoring NO-CDE formation or indirectly by allowing the development of nitrate-reducing bacterial species (Reed et al., 198 1; Kyrtopoulos et al., 1985). A short-term screening study of 6257 users of chlordiazepoxide, covering a follow- up period that was, at most, 7; years from the first recorded dispensing of the drug, showed no evidence of excess of any cancer (Fried- man and Ury, 1980). However, taking into account that the latency period for cancer development after exposure to carcinogens is often measured in decades from first exposure, the results of this analysis should be considered inadequate to provide a definitive proof of noncarcinogenicity in humans.

REFERENCES

ANDREWS. A. W., FORNWALD, J. A., AND LIJINSKY. W. ( 1980). Nitrosation and mutagenicity of some amine

drugs. Tovicol. Appl. Phurmacol. 52,237-244. BARTSCH, H., MALAVEILLE, C.. CAMUS, A.-M., MAR-

TEL-PLANCHE. G.. BRUN, G., HAUTEFEUILLE, A., SA-

BADIE, N.. BARBIN, A.. KUROKI, T.. DREVON, C., PIC- COLI, C., AND MONTESANO, R. (1980). Validation and comparative studies on 180 chemicals with S. typhimurium strains and V79 Chinese hamster cells in the presence of various metabolizing systems. Mzkzt. Res. 76, l-50.

BARTSCH, H., AND MONTESANO, R. (1984). Relevance

of nitrosamines to human cancer. Curcinogenesis 5, 1381-1393.

BLACKWELL. B. (1973). Psychotropic drugs in use today.

J. ‘4mer. Med. .4ssoc. 225, 1637- 164 1.

BRADLEY, M. 0.. BHUYAN. B., FRANCIS, M. C.. LANGENBACH, R., PETERSON, A., AND HUBERMAN. E. (198 1). Mutagenesis by chemical agents in V79 Chi-

nese hamster cells: A review and analysis of the litera- ture. A report of the Gene-Tox Program. Mutat. Res. 87,81-142.

BRAMBILLA, G. (1985). Genotoxic effects of drug/nitrite

interaction products: Evidence for the need of risk assessment. Pharmacol. Re.T. Commun. 17,307-321.

BRAMBILLA, G., CAJELLI, E., FINOLLO. R., MAURA. A..

PINO. A., AND ROBBIANO, L. (1985). Formation of DNA-damaging nitroso compounds by interaction of drugs with nitrite. A preliminary screening for detect-

ing potentially hazardous drugs. J. To-UCO/. Environ. Health 15, 1-24.

BRAMBILLA. G., CAVANNA, M., PARODI. S., SCIAB,& L.,

PINO, A., AND ROBBIANO, L. (1978). DNA damage in liver, colon, stomach, lung and kidney of BALB/c mice treated with I .2-dimethylhydrazine. Int. J. Can- cer 22, 174- 180.

CAJELLI, E.. CANONERO. R., MARTELLI, A., AND BRAM- BILLA. G. (1987). Methylglyoxal-induced mutation to 6-thioguanine resistance in V79 cells. Mutat. Res. 190,

47-50.

FRIEDMAN, G. D., AND URY, H. K. (1980). Initial screening for carcinogenicity ofcommonly used drugs.

J. Natl. Cancer Insr. 65,723-733. International Agency for Research on Cancer (IARC)

(1980a). IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals IO Humans, Vol. 24, Some Pharmaceutical Drugs, pp. 297-314. IARC, Lyon, France.

International Agency for Research on Cancer (IARC)

( 1980b). N-Nitroso Compounds: Analysis, Formation. and Occurrence. IARC Scientific Publication No. 3 1. IARC, Lyon, France.

KASTENBAUM. M. A., AND BOWMAN, K. 0. (1970). Ta-

bles for determining the statistical significance of mutation frequencies. Mutat. Res. 9.527-549.

KLEIN. D., GACONNET, N., POULLAIN, B., AND DERBY, G. ( 1978). Possibility of nitrosation during human di- gestion. I. Evaluation of precursor and affecting factors

in rlivo. II. Effect of food intake on the reaction. In L4RC Scientzjk Publications (E. A. Walker, M. Cas- tegnaro, L. Gricciute. and R. E. Lyle. Eds.). Vol. 19.

pp. 485-494. IARC. Lyon, France.

KOHN. K. W.. ERICKSON, L. C.. EWIG, R. A. G.. AND FRIEDMAN, C. A. (I 976). Fractionation of DNA from mammalian cells byalkalineelution. Biochemistry 15, 4629-4637.

KRAFF~, S., AND ONKEN, A. (1976). Induction of mitotic gene conversion in Saccharomvces cerevisiae with chlordiazepoxide and its N-nitroso derivative in a host-mediated assay. Mutat. Res. 34,333-336.

KYRTOPOULOS, S. A., DASKALAKIS, G.. LEGAKIS, N. I., KONIDARIS, N., PSARROU, E.. BONATSOS, G.. Go- LEMATIS. B.. LAKIOTIS, G., BLIOURAS. N.. AND OUT-


RAM, J. R. (1985). Studies in gastric carcinogenesis. II. Absence of elevated concentrations of N-nitroso compounds in the gastric juice of Greek hypochlorhydric individuals. Carcinogenesis 6, 1135-l 140.

LIJINSKY, W., AND TAYLOR, H. W. (1977). Nitrosa- mines and their precursors in foods. In “Origins OfHu- lnan Cancer” (H. H. Hiatt, J. D. Watson, and J. A. Winstein, Eds.). pp. 1579-1580. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.

MIRVISH, S. S. (1975). Formation of N-nitroso compounds: Chemistry, kinetics and in vivo occurrence. Toxicol. Appl. Pharmacol. 31,325-35 1.

MYHR, B. C., AND DI PAOLO, J. A. (1978). Mutagenesis by N-acetoxy-2-acetyl aminofluorene of Chinese hamster V79 cells is unaffected by caffeine. Chem. Biol. In- teract. 21, l-18.

PREDA. N., POPA, L.. AND GALEA. V. (1976). N-Nitroso compound formation by chlordiazepoxide and nitrite interaction in vitro and in vivo: Protective action of ascorbic acid. In Environmental N-Nitroso Com- pounds Analysis and Formation, IARC Scientific Pub- lication No. 14. pp. 301-3 14. IARC, Lyon, France.

RAISFELD-DANSE, I. H., AND CHEN, J. (1983). Drug in- teractions. III. Formation of nitrosamines from therapeutic drugs. Formation, mutagenic properties and safety assessment of propranolol hydrochloride with respect to the intragastric formation of N-nitrosopro- pranolol under conditions found in patients. J. Phar- macol. Exp. Ther. 225,7 13-7 19.

REED, P. I.. HAINES, K., SMITH, P. L. R., AND HOUSE. F. R. (1981). Effect of cimetidine on gastric juice N- nitrosamine concentration. Lancet 2,553-555.

ROPER, P. R.. AND DREWINKO, B. (1976). Comparison of in vitro methods to determine drug-induced cell le- thality. Cancer Res. 36,2 182-2 188.

R~~MKE, C. L.. AND DE JONGE, H. (1964). Design, statistical analysis and interpretation. In Evaluation qfDrug Activities: Pharmacometrics (D. R. Laurence and A. L. Bacharach, Eds.). Vol. 1, pp. 80-82. Academic Press, New York.

SINA, J. F., BEAN, C. L., DYSART, G. R., TAYLOR, V. I.. AND BRADLEY, M. 0. (1983). Evaluation of the alkaline elution/rat hepatocyte assay as a predictor ofcarci- nogenic/mutagenic potential. Mutat. Res. 113, 357-

391. STROM, S. C.. JIRTLE. R. L., JONES, R. S.. NOVICKI,

D. L., ROSENBERG, M. R.. NOVOTNY, A., IRONS, G..

MCLAIN, J. R., AND MICHALOPOULOS, G. (1982). Iso- lation, culture, and transplantation of human hepatocytes. J. Natl. Cancer Inst. 68,77 l-778.

STROM, S. C., NOVICKI, D. L.. NOVOTNY, A., JIRTLE, R. L., AND MICHALOPOULOS, G. (1983). Human hepatocyte-mediated mutagenesis and DNA repair activity. Carcinogenesis 4,683-686.

SWENBERG. J. A., PETZOLD, G. L., AND HARBACK, P. R. (1976). In vitro DNA damage/alkaline elution assay for predicting carcinogenic potential. Biochem. Bio- ph.vs. Res. Commun. 12,732-738.

TAKEDA. Y., AND KANAYA, H. (1981). Formation of nitroso compounds and mutagens from tranquilizers by drug/nitrite interaction. Cancer Lett. 12,8 l-86.

TANNENBAUM, S. R., ARCHER. M. C.. WISHNOK, J. S., CORREA. P.. CUELLO, C., AND HAENSZEL, W. (1977). Nitrate and etiology ofgastric cancer. In Origins ofHu- man Cancer (H. H. Hiatt, J. D. Watson, and J. A. Winstein. Eds.), pp. 1609- 1625. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.

TANNENBAUM, S. R.. MORAN, D., RAND, W., CUELLO, C., AND CORREA, P. (1979). Gastric cancer in Colum- bia. IV. Nitrite and other ions in gastric contents of residents from high-risk region. J. Natl. Cancer Inst. 62,9- 12.

TERRACINI, B., MAGEE. P. N., AND BARNES, J. M. (1967). Hepatic pathology in rats on low dietary levels of dimethylnitrosamine. Brit. J. Cuncer 21,559-565.

WALSER. A., FRYER. R. I., STERNBACH, L. H.. AND ARCHER, M. ( 1974). Quinazolines and 1,4-benzodiaz- epines. LXV ( I ) Some transformations of chlordiazepoxide. J. Heterocycl. Chem. 11,6 19-62 1.

WALTERS, C. L. (1980). The exposure of humans to nitrite. Oncology31,289-296.

WILLIAMS. G. M. (1977). The detection of chemical carcinogens by unscheduled DNA synthesis in rat liver primary cell cultures. Cancer Res. 37, 1845- 185 1,

WILLIAMS, G. M., LASPIA, M. F.. AND DUNKEL, V. C. (1982). Reliability of the hepatocyte primary culture/ DNA repair test in testing of coded carcinogens and noncarcinogens. Mutat. Res. 97,359-370.

WILLIAMS, J. R., LITTLE, J. B., AND SHIPLEY, W. U. ( 1974). Association of mammalian cell death with a specific endonucleolytic degradation of DNA. Nature (London) 252,754-755.

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