genotoxic activities of drug-nitrite interaction products

©2003 Marcel Dekker, Inc. All rights reserved. This material may not be used or reproduced in any form without the express written permission of Marcel Dekker, Inc.

MARCEL DEKKER, INC. • 270 MADISON AVENUE • NEW YORK, NY 10016

DRUG AND CHEMICAL TOXICOLOGY

Vol. 26, No. 4, pp. 295–308, 2003

Genotoxic Activities of Drug-Nitrite Interaction Products

Gul Ozhan and Buket Alpertunga*

Department of Pharmaceutical Toxicology, Faculty of Pharmacy,

Istanbul University, Istanbul, Turkey

ABSTRACT

At first 28 orally administered drugs, considered to be potentially nitrosatable on

the basis of their chemical structure, have been nitrosated with nitrite under

simulated stomach conditions. A maximum daily dose of each drug was

incubated with a nitrite concentration that can be found after a normal meal in

the stomach at 37�C over 1 h. Reaction was started at pH 6.8–7.0 and stopped

at pH 2.0, so we had the same pH change that occurs in the stomach. Secondly

the genotoxic activities of drug-nitrite interaction products were tested by the

umu-test with Salmonella typhimurium TA 1535/pSK1002 as tester strain in

the presence and absence of metabolic activation. By the umu-test, among the

nitrosation products of drugs examined 22 products showed genotoxicity at

different levels. Other six products showed negative results by the umu-test.

Key Words: Drug-nitrite interaction; Umu-test; N-nitrosamines; Genotoxicity.

*Correspondence: Dr. Buket Alpertunga, Department of Pharmaceutical Toxicology,

Faculty of Pharmacy, Istanbul University, 34116 Istanbul, Turkey; Fax: þ90 212 519 08 12;

E-mail: [email protected].

295

DOI: 10.1081/DCT-120024844 0148-0545 (Print); 1525-6014 (Online)

Copyright & 2003 by Marcel Dekker, Inc. www.dekker.com

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INTRODUCTION

N-Nitroso compounds (NOCs) represent a major class of important chemicalmutagens, carcinogens, teratogens, and immunotoxic agents, which have beendescribed as a serious hazard to human health.

NOCs have been shown to occur in food products, water, alcoholic beverages,industrial products, agricultural products, cosmetics, drugs, tobacco products, andhuman biological fluids.[1–3] The most important property of these compounds isthat they exist endogenously. Drugs contain nitrosatable groups such as secondary,tertiary amines, and amides that can produce NOCs by interaction with nitrite.Stomach provides a suitable medium with its low pH degree for the interactionwith nitrite. Many of these drugs have been subjected to the Nitrosation AssayProcedure (NAP test), a reference procedure to determine the nitrosatability ofdrugs, recommended by WHO with a molar excess of nitrite (4:1) at pH 3–4.[4,5]

Most of them were converted into their N-nitroso derivatives, and have also beentested for carcinogenic activities.[6]

Bacterial short-term tests are now widely used to detect potential carcinogensand mutagens. Among them, the umu-test system, developed by Oda et al.,[7] is asimple, sensitive, and rapid bacterial colorimetric assay to evaluate the genotoxicactivities of the chemicals and is statically equivalent to the Ames test which is themost popular of the bacterial assays.[8,9]

In this study, the nitrosation products of 28 drugs formed by the incubation withnitrite under simulated stomach conditions were tested for their genotoxic effects bythe umu-test with and without metabolic activation.

EXPERIMENTAL

Chemicals and Reagents

The enzyme substrate o-nitrophenyl-b-D-galacto-pyranoside (ONPG), 4-nitro-quinoline N-oxide (NQO), 4-nitro-o-phenylenediamine (NODP), 2-aminoanthracene(2-AA), 9,10-dimethyl-1,2-benzanthracene (DMBA), and b-nicotinamide adeninedinucleotide phosphate (NADP) were from Sigma. Bacto tryptone and yeast extractwas from Difco. All the other chemicals were from Merck. The pharmaceuticalpreparations were purchased from the manufacturers. N-Nitrosomaprotiline wasprepared from maprotiline hydrochloride in our laboratory by the method describedby Alpertunga and Mes� eci.[10]

Umu-Test Principle

The principle of the umu-test is based on the ability of the DNA-damagingagents to induce the umu operon. A plasmid (pSK 1002) carrying a fused geneumuC’-’lacZ was introduced into Salmonella typhimurium TA 1535. The strain TA1535/pSK 1002 (kindly provided by Dr. Y. Oda of Osaka Prefectural Institute of

296 Ozhan and Alpertunga

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Public Health) enabled us to monitor the levels of umu operon expression bymeasuring the cellular b-galactosidase activity produced by the fusion gene.[7]

Overnight Culture Growth

The bacteria were grown in Luria broth (LB medium) supplemented with20 mg/mL ampicillin. The overnight culture of the tester bacterial strain wasdiluted 20-fold with tryptone glucose ampicillin broth (TGA medium) and wasincubated at 37�C until the bacterial density reached an absorbance of 0.25–0.30at 600 nm.

Media, Buffer, and Solution

LB medium: Bacto tryptone 10 g, yeast extract 5 g, NaCl 5 g/L of distilled water.LB medium is supplemented with 20 mg/mL ampicillin.

TGA medium: Bacto tryptone 10 g, NaCl 5 g, glucose 2 g/L of distilled water.Z buffer: NaH2PO4�2H2O 6.2 g, Na2HPO4�7H2O 16.1 g, KCl 0.75 g,

MgSO4�7H2O 0.25 g, Sodium dodecyl sulfate 1 g, b-mercaptoethanol 2.7mL/L ofdistilled water and adjusted to pH 7.

ONPG solution: 400mg ONPG per 100mL of phosphate buffer pH 7.Compounds to be tested (NQO,NODP, 2-AA,DMBA)were dissolved inDMSO.

Activation Mixture

The S9 microsome fraction was prepared from livers of male Sprague Dawleyrats pretreated with phenobarbital. The activation mixture was prepared accordingto Ames et al.[11]

The S9 mix contained per 10mL: 3mL of S9 fraction, 1mL of 0.04M NADP,0.25mL of 0.2M G6P, 0.32mL of 0.25M MgCl2�6H2O, 0.33mL of 1M KCl, 5mLof 0.2M NaHPO4, 0.1mL of distilled water.

Nitrosation

Nitrosation reactions were carried out essentially as described by Ziebarth andTeichmann.[12] A maximum daily dose of each drug was dissolved in 500mL ofdistilled water. The solution was adjusted to pH 6.8–7.0 with sodium hydroxideand incubated with 0.1mL of 0.64mol/L aqueous sodium nitrite. All incubationswere carried out in the dark at 37�C with shaking for 60min to approximate con-ditions present in the stomach. Every 5min, �50 mL dilute hydrochloride acid wasadded in order to gradually adjust the pH of the mixture from 6.8 to 2 over theperiod of 60min. The reactions were terminated by the addition of excess sulfamicacid. The incubation mixtures were extracted several times with dichlormethane. Thepooled organic phases were dried with Na2SO4 and evaporated under reduced

Drug-Nitrite Interaction Products 297

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pressure. The residues were dissolved and diluted in 1–2mL DMSO according totheir solubility and 0.05mL of DMSO extracts was assayed for genotoxicity by theumu-test.

On the other hand, a maximum daily dose of each drug was incubated withoutsodium nitrite under the given reaction conditions. As a control aqueous sodiumnitrite was treated as the sample in the nitrosation reaction. The genotoxic activitiesof these reaction products were assayed by the umu-test as well.

Umu-Test Procedure

The umu-test was performed according to the methods of Oda et al.[7] Thebacterial culture was subdivided into 0.75mL portions in tubes and 0.05mL ofDMSO extracts was added to each tube. For some drugs, 0.05mL of DMSO extractsshowed killing effects to the tester strain. So, appropriate dilutions of the DMSOextracts were used. Then, either 0.2mL of 0.1M phosphate buffer pH 7.4 or S9mix was added. After 2 h of incubation at 37�C, the bacterial density wasmeasured at 600 nm (OD600) by Shimadzu Model 1201 Spectrophotometer. Thelevel of b-galactosidase activity (units, U) was measured by the method of Milleras follows[13]:

Fractions (0.1mL) of the culture were diluted with 0.9mL of Z buffer andmixing vigorously. The enzyme reaction was initiated by the addition of 0.1mLONPG solution at 37�C. After 15min, the reaction was stopped by adding 0.5mL1M Na2CO3 and the absorbance of the mixture was measured at 420 nm (OD420).In addition, each sample was measured at 550 nm (OD550) in order to correct forlight scattering due to cell fragments. The b-galactosidase activity is calculatedaccording to the formula of Miller.

b-Galactosidase activity (U) ¼1000ðOD420 � 1:75OD550Þ

t: v:OD600

t: time of the reaction in minutesv: volume of culture in the assay, in mL

In the umu-test, more than two-fold increase in b-galactosidase activity abovethe background level (DMSO) will be respected as positive response.

The umu-test procedure was applied to four positive controls, directly actingmutagens (4-nitroquinoline N-oxide¼NQO; 4-nitro-o-phenylenediamine¼NODP)and promutagens (2-aminoanthracene¼ 2-AA; 9,10-dimethyl-1, 2-benzanthracene¼DMBA) before evaluation of the genotoxic activities of the nitrosation products.

RESULTS AND DISCUSSION

The aim of this study is to evaluate the genotoxic potency of drug/nitrite inter-action products formed under simulated stomach conditions. The chemical formulaeof the drugs examined are listed in Table 1. These compounds were selected, for


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Table 1. Drugs used in the experiment.

Generic name Formula

A daily max. dose

mg mmol

Cimetidine H2 receptor antagonist

N N

H3C CH2 S CH2CH2NHCNH

NHCH3

C N

H

800 3.17

Ranitidine H2 receptor antagonistO CH2SCH2CH2NHCH2 C

CHNO2

NHCH3N

CH3

CH3

300 0.95

Acebutolol b-adrenergic antagonist

COCH3

OCH2CHCH2-NH-CH(CH3)2

NHCO(CH2)2CH3

OH

300 0.89

Atenolol b-adrenergic antagonist CH2CONH2

OCH2CHCH2NHCH(CH3)2

OH

100 0.38

Sotalol b-adrenergic antagonist

CH CH2 NHCH (CH3)2

OH

NHSO2CH3 240 0.88

Metoprolol b-adrenergic antagonist

OCH2CHCH2NHCH(CH3)2

CH2CH2OCH3

OH

200 0.75

Propranolol b-adrenergic antagonist OCH2CH

OH

CH2NHCH(CH3)2 20 0.08

Betahistine histamine analogous N CH2CH2NHCH3 16 0.12

Nifedipine calcium channel blocker

N CH3H3C

COOCH3

NO2

H3COOC

H

40 0.12

Amlodipine calcium channel blocker

HN CH2OCH2CH2NH2H3C

COOC2H5

CI

H3COOC

10 0.03

Bromazepam tranquilizer

N

N

Br

O

N

H 3 9.49� 10�3

Chlordiazepoxide tranquilizer

N

NNHCH3

C6H5

CIO

40 0.13

Opipramol antidepressantN

CH2CH2CH2 CH2CH2OHN N 100 0.28

(continued )


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Table 1. Continued.

Generic name Formula

A daily max. dose

mg mmol

N-Nitrosomaprotiline CH2CH2CH 2 NNO

C H 3

Paroxetine (antidepressant) N

OCH2O

O

F

H50 0.15

Sertraline (antidepressant)

CICI

NH CH3 50 0.16

Amineptine (antidepressant)

NH-(CH2)6-COOH

100 0.30

Fluoxetine (antidepressant) F3C O CHCH2CH2NHCH3 20 0.07

Ephedrine (�þ b-adrenergic antagonist)CH CH CH3

NH CH3OH 0.05 0.3�10�3

Pseudoephedrine (�þ b-adrenergic antagonist)CH CH CH3

NH CH3OH 120 0.73

Ethambutol (tuberculostatic)CH NH

HOH2C

H5C2

(CH2)2 NH CHCH2OH

C2H5

1,500 7.34

Ritodrine (tocolytic)HO CH CH

CH3

OH

NHCH2CH2 OH10 0.04

Terbutaline (b-adrenergic antagonist)CH CH2NHC(CH3)3

OHHO

HO

5 0.02

Salbutamol (b-adrenergic antagonist)HO

HOCH2

CHCH2NHC(CH3)3

OH 8 0.03

Ambroxol (mucolytic)CH2 NH OH

NH2Br

Br

30 0.08

Enalapril (ACE inhibitor)CH2CH2 C NH C C

COOC2H5

H

N

COOHCH3

OH

40 0.11

Tizanidine (muscle relaxant)

NH

N NH

N

SNCI

12 0.05

Astemizole (antihistaminic)

NH

N

CH2 F

NH N CH2CH2 OCH3

10 0.02


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Table 2. Results of the umu-test on the genotoxic activity of drug-nitrite interaction

products.

Compound

Concentration*

(mg/mL)

b-Galactosidase

activitya (U)**

Induction

factor (I )***

�S9 þS9 �S9 þS9

Controlb 139±26 102±21 — —

NQO 0.001 620±23 — 4.5 —

NODP 0.040 541±25 — 3.9 —

2-AA 0.004 — 492±9 — 4.8

DMBA 0.010 — 302±17 — 3.0

Sodium nitrite 1.00 139±6 82±7 1.0 0.8

Cimetidine 3.20 125±10 — 0.9 —

6.40 195±6 214±30 1.4 2.1

9.60 236±6 — 1.7 —

12.80 292±13 224±17 2.1 2.2

16.00 209±19 224±2 1.5 2.2

Ranitidine 4.00 164±6 493±45 1.2 4.8

6.00 146±5 528±12 1.1 5.2

8.00 172±13 453±6 1.2 4.4

Acebutolol 4.20 205±1 139±1 1.5 1.4

8.40 307±2 217±2 2.2 2.1

12.60 427±3 319±3 3.1 3.1

Atenolol 0.70 209±8 275±25 1.5 2.7

1.40 292±4 337±7 2.1 3.3

2.10 320±10 245±5 2.3 2.4

2.80 500±3 214±18 3.6 2.1

3.50 403±23 204±10 2.9 2.0

Sotalol 3.00 253±22 172±5 1.8 1.7

6.00 354±3 250±11 2.5 2.4

9.00 349±14 297±8 2.5 2.9

Metoprolol 0.60 299±1 207±9 2.1 2.0

1.20 271±11 171±11 1.9 1.7

Propranolol 0.08 431±10 255±3 3.1 2.5

0.16 473±13 377±6 3.4 3.7

0.32 639±42 469±12 4.6 4.6

0.48 473±15 347±20 3.4 3.4

0.64 320±25 326±9 2.3 3.2

0.72 236±3 265±3 1.7 2.6

Betahistine 0.20 292±14 286±3 2.1 2.8

0.30 403±24 306±3 2.9 3.0

0.40 528±37 377±4 3.8 3.7

0.50 570±26 347±3 4.1 3.4

0.60 445±43 296±3 3.2 2.9

Nifedipine 0.07 360±22 289±16 2.6 2.8

0.15 521±14 373±34 3.7 3.6

(continued )


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Table 2. Continued.

Compound

Concentration*

(mg/mL)

b-Galactosidase

activitya (U)**

Induction

factor (I )***


Amlodipine 0.06 223±11 171±20 1.6 1.7

0.12 182±22 223±8 1.3 2.2

Bromazepam 0.75 232±12 192±6 1.7 1.9

1.50 450±18 213±6 3.2 2.1

Chlordiazepoxide 0.005 297±17 310±14 2.1 3.0

0.010 360±36 462±26 2.6 4.5

Opipramol 0.05 528±15 191±7 3.8 1.9

0.10 664±8 215±7 4.8 2.1

N-Nitrosomaprotiline 0.038 — 68±16 — 0.7

0.076 376±12 257±24 2.7 2.5

Paroxetine 0.70 304±16 309±8 2.2 3.0

1.40 391±4 358±2 2.8 3.5

2.10 450±9 309±8 3.2 3.0

Sertraline 0.07 179±14 145±2 1.3 1.4

0.14 222±6 102±10 1.6 1.0

Amineptine 1.40 270±19 128±5 1.9 1.2

2.80 267±33 226±7 1.9 2.2

Fluoxetine 0.06 351±29 337±7 2.5 3.3

0.09 506±17 441±22 3.6 4.3

0.12 318±4 306±11 2.3 3.0

Ephedrine 1.00 358±32 237±14 2.6 2.3

2.00 718±19 510±16 5.2 5.0

Pseudoephedrine 0.84 153±3 122±10 1.1 1.2

1.68 459±17 347±13 3.3 3.4

2.52 389±24 377±10 2.8 3.7

3.36 514±7 408±10 3.7 4.0

4.20 695±47 439±5 5.0 4.3

Ethambutol 25.00 213±26 115±2 1.5 1.1

50.00 190±5 131±5 1.4 1.3

75.00 199±7 149±4 1.4 1.5

Ritodrine 0.14 165±29 143±15 1.2 1.4

0.28 181±8 156±4 1.3 1.5

0.42 131±10 211±6 0.9 2.1

Terbutaline 0.04 292±19 122±3 2.1 1.2

0.08 570±9 184±4 4.1 1.8

0.12 487±21 214±18 3.5 2.1

Salbutamol 0.08 196±29 258±2 1.4 2.5

0.16 150±17 305±7 1.1 3.0

Ambroxol 0.24 320±15 180±18 2.3 1.8

0.48 306±34 221±7 2.2 2.2

Enalapril 0.56 168±5 130±9 1.2 1.3

1.12 402±3 361±3 2.9 3.5

1.66 291±2 278±17 2.1 2.7

(continued )


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they are commonly administered by the oral route and assessed as being nitrosatableby their chemical structure. The genotoxic activities of drug/nitrite interactionproducts were investigated by the umu-test, which is a colorimetric assay based onthe production of b-galactosidase as a response to DNA damage.

At first the maximum daily doses of the selected drugs were incubated with anitrite concentration that can be found after a normal meal in the stomach at 37�Cover 1 h.[14] Reaction was started at pH 6.8–7.0 and stopped at pH 2.0 attending theshifts of pH occurring in the course of the digestive process. Secondly the genotoxicactivities of drug/nitrite interaction products were investigated by the umu-test withand without metabolic activation (�S9). The results are summarized in Table 2.As shown in Table 2, 22 of the compounds showed genotoxic activities in theumu-test at different levels after nitrosation. The nitrosation products of cimetidine,ephedrine, pseudoephedrine, betahistine, propranolol, sotalol, atenolol, acebutolol,nifedipine, enalapril, tizanidine, paroxetine, chlordiazepoxide, fluoxetine, terbutaline,and n-nitrosomaprotiline showed positive genotoxicity independent of themetabolic activation system (�S9). Ranitidine and salbutamol were positive onlyin the presence of S9 mixture (þS9), while ambroxol, astemizole, opipramol, andbromazepam were positive in the absence of S9 mixture (�S9). The drugs alone aretested in the same order and found to be inactive by the umu-test in the absence andin the presence of metabolic activation system.

As shown in Table 2 for the majority of drugs examined, the genotoxicity of thenitrosation mixture was not affected by S9 mixture containing liver preparation,neither in the sense of activation nor deactivation. This implies that the formationsof direct-acting products are rather frequent. The induction factors indicated inTable 2 shown that a dose response relation does not occur, because of the bacterialactivity of the nitrosation mixtures at higher level to the tester strain.

Table 2. Continued.

Compound

Concentration*

(mg/mL)

b-Galactosidase

activitya (U)**

Induction

factor (I )***


Tizanidine 0.04 292±12 525±8 2.1 5.1

0.12 356±17 611±8 2.6 6.0

0.16 367±10 751±8 2.6 7.4

Astemizole 0.14 301±18 190±22 2.2 1.9

0.28 305±9 201±18 2.2 2.0

*The amount of the drug, which is found in the incubation mixture by the umu-test.

** amean±SD (n¼ 4).bsolvent control, DMSO (50 mL per assay).

***The ‘‘induction factors’’ for a given concentration I were calculated by dividing the

b-galactosidase activity Uc at a given concentration (c) with the background b-galactosidaseactivity Uo, I¼Uc/Uo. The inducibility of these compounds was divided into three classes.

The potent inducers, intermediate inducers, and weak inducers, which induced umu gene

expression 6-fold, 3-fold, and 2-fold over the background level, respectively.


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In many cases drug/nitrite interaction yields the expected N-nitroso derivative;e.g., N-nitrosoephedrine,[15] N-nitrosochlordiazepoxide.[16] Several of these N-nitrosoderivatives were tested for mutagenicity in the Salmonella/microsome reversion testand for the capability of inducing DNA fragmentation in CHO cells. For a limitednumber of drugs carcinogenicity tests have been performed in rodents by long-termadministration of the nitroso derivative.[17–19]

Among the compounds investigated in this study cimetidine is the drug for whichthe interaction with nitrite is exhaustively examined. The histamine H2-receptorantagonist cimetidine has been shown to react with nitrite to produce nitroso deriva-tives, mononitrosocimetidine, and dinitrosocimetidine.[20,21] The major product,mononitrosocimetidine has been shown to have the methylating ability comparableto that of the known gastric carcinogen, N-methyl-N 0-nitro-N-nitrosoguanidine(MNNG).[21,22] Several studies demonstrated the genotoxicity of nitrosatedcimetidine, using a variety of short-term test systems with different biologicalend-points. Nitrosocimetidine was found to produce point mutations in bacteriahis-Salmonella typhimurium both in the presence and absence of S9 liver preparationsand to induce DNA damage in trp-Escherichia coli.[23,24] But no treatment-relatedtumors were found in male and female rats given nitrosocimetidine in the drinkingwater for more than two years.[25] On the other hand a mutagenic response wasobtained in the Salmonella/microsome reversion test by preincubating sodium nitriteand cimetidine in equimolar amounts (about 80 mmol per plate) in human gastric juicefrom untreated individuals or by adding nitrite to gastric juice samples from patientsreceiving cimetidine.[26] By the umu-test the nitrosation mixture of cimetidine showeda weak genotoxic activity (Imax¼ 2.2) with and without S9. The other histamineH2-receptor antagonist ranitidine showed moderate activity (Imax¼ 5.2) in thepresence of S9. De Flora et al. found ranitidine to be nonmutagenic. However, ascimetidine, after preincubation with nitrite in human gastric juice from untreatedindividuals or simply acidification of nitrite–ranitidine mixtures they found thatsome mutagenicity resulted especially in the presence of liver preparations accordwith our findings.[27] On the other hand these in vitro findings are in contrast to thereported that no mutagenic nitrosation product of ranitidine is to be formed in manunder any conceivable physiological conditions.[28]

b-Adrenergic-blocking agents are potentially nitrosatable secondary amines,which are widely used for the treatment of hypertension and cardiac arrhythmias.Their N-nitroso derivatives have been already synthesized and characterized,[29,30]

but there are only few studies concerning their genotoxicity. Robbiano et al. reportedthat N-nitroso derivatives of six b-blocking agents produce dose-dependent DNAfragmentation and DNA repair synthesis in both rat and human hepatocytes atsubtoxic concentrations.[30] Synthetic N-nitrosopropanol has been found to be inac-tive in the Ames test and in the hepatocyte-mediated mammalian cell mutagenesisassay.[31] We found that the nitrosation mixture of the b-blocking agents studiedexcept metoprolol showed weak to intermediate induction in the umu-test systemindependent of the metabolic activation system (�S9).

Most of the tranquilizers contain nitrosatable groups. In the umu-test system thenitrosation mixtures of opipramol and bromazepam showed intermediateinduction (Imax¼ 4.8; Imax¼ 3.2) without metabolic activation, while the nitrosationmixture of chlordiazepoxide showed weak induction in the absence of S9 mix


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and increasing induction in the presence of S9 mix. For the chlordiazepoxide highmutagenicity (�S9) was found in the Ames test using Salmonella typhimurium TA98and TA100 as tester strains after the nitrosation with excess of nitrite (drug/nitrite molratio 1:10).[32] In the same study bromazepam gave moderately mutagen product(s)while opipramol gave highly mutagen product(s) without S9. If the drug reacted with alow concentration of nitrite (3:1) a weak mutagenicity (�S9) was observed by theAmes assay.[17] N-Nitrosochlordiazepoxide or chlordiazepoxide plus sodium nitritehave been shown to induce DNA damage in rats by oral administration.[33]

The nitroso derivatives formed from ephedrine[14] and ethambutol[34] with excessof nitrite have been identified. N-Nitrosoephedrine (NEP) showed positive results insome short-term tests.[35] In the Ames assay NEP showed significant mutagenicactivity in the presence of S9 mix.[36] In our study, however, the nitrosation mixtureof ephedrine and pseudoephedrine induced umu-gene expression (Imax¼ 5.2;Imax¼ 5.0) independent of the metabolic activation system (�S9). The nitrosationmixture of ethambutol was inactive in the umu-test system, in agreement with thereported Ames test result.[37]

Except for the drugs mentioned above, there is no information about the nitrosoderivative and their genotoxic activities of the remaining compounds in the study.

We have previously reported that maprotiline, an antidepressant drug,reacts with nitrite under simulated stomach conditions produce the correspondingN-nitrosomaprotiline (NOM).[10] In the umu-test system an intermediate genotoxicactivity was observed with synthetic NOM independent of the metabolic activationsystem (�S9).

This study was carried out to assess whether the nitrosatable drugs give riseto hazardous amounts of their genotoxic nitroso derivative(s) produced in anenvironment resembling the gastric juice. It is very difficult to estimate the extentto which the nitrosation take place in human and the amounts of N-nitroso deriva-tive, because the nitrosation depends on various factors, such as the concentration ofdrug and nitrite, pH, pKa of drugs, stomach contents, metabolic activation system,and concentration of catalysts and inhibitors. For most drugs, the reaction withnitrite is complex, giving rise to more than one nitrosamine product. On the otherhand, there is no relationship between the chemical structures of the drugs and theirgenotoxic activities by umu-test. For example terbutaline and salbutamol, whichhave similar structure and close pKa values, showed different activities in theumu-test system. The variation of the genotoxic potency of the nitrosation productssuggest various reaction products are formed after interaction with nitrite undersimulated stomach conditions.

Although the examined drugs have been used for several years on patientswithout problem, the result of this study needs attention, because even the verysmall amounts of N-nitroso products formed in the stomach represent an increasein exposure to genotoxic chemicals. In order to get an accurate estimate of actualrisk of nitrosation of these drugs, it must be known the structure and amount of theN-nitroso derivatives formed in human stomach, as well as their pharmacokineticbehavior, stability, concentration reaches the target cells and carcinogenic–mutagenic potency.

The isolation and identification of N-nitroso derivative(s) of examined drugs arebeing currently under examination.


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ACKNOWLEDGMENT

This work was supported by the Research Fund of the Istanbul University(Project numbers: T-459/071197 B-566/17072000).

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genotoxic activities of drug-nitrite interaction products

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