Urinary excretion of S-benzylmercapturic acid as an indicator of N-nitroso-N-methylbenzylamine exposure

Cancer Letters, 57 (1991) 193- 198 Elsevier Scientific Publishers Ireland Ltd.

193

Urinary excretion of S-benzylmercapturic acid as an indicator of IV-nitroso-N-methylbenzylamine exposure D.-X.

C. Malaveillea,

Lina3b, M. Friesen”,

“International

D.E.G.

for Research on Cancer, 150 Cows Albert Fujian Medical College, Fuzhou (P.R. China)

Agency

and Food

Hygiene,

(Received (Accepted

28 January 2 February

chromatography-mass

gas

(GC-MS). The identity of the urine was confirmed by full scan GC-MS. The amount of urinary SBzMA varied with the dose of NMBzA (up to 5 mg/kg) and

spectrometry SBzMA in

with

rat

strain.

vestigated,

For

most

of

the a

three

2.5

strains

mg/kg

dose

inof

SBzMA was excreted within 24 h. Comparison of the levels of this SBzMA excreted by rats treated with equivalent doses of either NMBzA or benzaldehyde

indicates

that urinary

SBzMA

is derived mainly from benzylating species resulting from the hydroxylation of the methyl

of NMBzA.

group

Keywords: S-Benzylmercapturic acid; N-nitroso-N-methylbenzylamine, urine; chromatography-mass spectrometry

rat; gas

Materials

Introduction N-Nitroso-N-methylbenzylamine Correspondence to: H. Bartch. International Research on Cancer, 150 Cours Albert Thomas, Cedex

Published

and Printed

Lyon (France)

and bDivision

of Nutrition

0

Agency for 69372 Lyon.

1991 Elsevier Scientific

in Ireland

Publishers

and Methods

Chemicals NMBzA was kindly provided by Dr R. Preussman (German Cancer Research Center, Institute of Toxicology and Chemotherapy, F.R.G.). SBzMA was purchased from Janssen

(NMBzA)

08. France.

0304-3835/91/$03.50

H. Bartscha

is a potent esophageal carcinogen in experimental animals [Z]. Several investigations carried out in China suggest that subjects living in a high-risk area for the disease are exposed to this carcinogen [9,11,12]. NMBzA is activated in vitro through a-hydroxylation by cytochrome P-450 to generate either methylating or benzylating species [6,16]. In a previous study [lo], we found that in bacteria, DNA benzylation contributed predominantly to the mutagenicity of NMBzA in the presence of a rat liver metabolic activation system. Furthermore, in rats, conjugation with glutathione (GSH) was shown to inactivate the benzylating species, with high interstrain variability [lo]. As a follow-up to that study, we have developed a gas chromatography-mass spectrometry (GC-MS) assay to monitor human exposure to NMBzA based on urinary excretion of S-benzylmercapturic acid (SBzMA)We have examined the biological relevance of this indicator in several strains of rats with different metabolic activation and/or conjugation capacities.

The excretion of S-benzylmercapturic acid (SBzMA) in the urine of rats treated with Nnitroso-N-methylbenzylamine (NMBzA) was by

Thomas,

and

1991) 1991)

Summary

determined

Shuker”

Ireland

Ltd

194

Chimica (Belgium). Benzaldehyde and d7benzyl chloride were obtained from AldrichChemie (F.R.G.). N-Acetylcysteine was from Sigma Chemical Co. (St Louis, MO, U.S.A.). N-Methyl-N-nitroso-4-toluenesulfonamide was obtained from Merck-Schuchardt (F.R.G.). All other chemicals used were analytical grade. Synthesis of internal standard S-(d7-benzyl)mercapturic acid d7-Benzyl chloride (133.6 my, 1 mmol) in NJ-dimethylformamide (3 ml) was treated with ZV-acetylcysteine (163.2 mg, 1 mmol) in water (3.5 ml) containing sodium carbonate (106 mg, 1 mmol) and the mixture was stirred at room temperature for 4 h. The mixture was diluted with water (10 ml) and extracted with diethyl ether (3 x 5 ml). The aqueous phase was adjusted to pH 3 and extracted with chloroform (3 x 10 ml). The organic solvent was dried with Na2S04 and evaporated to give a colorless oil. Trituration with a small quantity of ether led to crystallization. After cooling, the crystals were filtered and washed with cold ether. The yield was 108 mg. Animals Male Sprague-Dawley, Wistar and Fischer 344 rats were purchased from Iffa-Credo (St Germain sur Arbresle, France) ; BD VI rats were bred in the IARC laboratory. All rats used were 8-9 weeks old. Animals were housed in a controlled environment and maintained on a Charles River CRF diet and tap water ad libitum. After overnight fasting, 16 rats of each strain were divided into four dose groups. Animals of each group were intragastrically administered 0, 1.5 (O.Ol), 2.5 (0.017) and 5 mg/kg (0.034 mmol) NMBzA, respectively. The treated animals were kept individually in metabolic cages. Urine samples were collected in 50-ml polyethylene bottles during 24 h after treatment. For kinetic studies, the urine samples from the rats treated with 5 mg/kg NMBzA were collected during 24 h by 6- or 12-h intervals. Two groups of four SpragueDawley rats were intragastrically dosed with

0.017 and 0.034 mmol/kg benzaldehyde, respectively. NMBzA was dissolved in olive oil and benzaldehyde in demineralized water. Control animals were treated with vehicle only. Urine samples were stored for less than 1 month at -20°C before analysis. Identification and quantitative determination of SBzMA Urine sample preparation. Urinary extracts containing SBzMA were prepared according to a modification of the method described by Norstram et al. [13]. Briefly, aliquots of rat urine spiked with 200 ng d7-SBzMA (internal standard) were acidified with concentrated HCI to pH 1 and then slowly percolated through Cl8 Sep-Pak cartridges (Waters Associates, Milford, MA, U.S.A.). The cartridges were washed successively with 0.01 M HCl (10 ml) and benzene (1 ml). After as much solvent as possible was removed by blowing air through the cartridges, SBzMA was eluted with 2 ml of chloroform/acetic acid (99: 1 v/v). The eluate was collected and taken to dryness by vacuum centrifugation. The residue was taken up in 0.5 ml ether and derivatized with diazomethane derived from Nmethyl-N-nitroso-4-toluenesulfonamide. After evaporation of the solvent, the methyl ester was dissolved in 10 ~1 ethyl acetate for GC-MS analysis. Gas chromatography-mass spectrometry. Analyses were performed on a Hewlett Packard 5970A mass selective detector operated in the electron impact mode at 70 eV coupled through a heated interface (260°C) to a Hewlett Packard 5980 GC. The GC was equipped with a HP Ultra-2 fused silica capillary column (25 m x 0.2 mm i.d., 0.33 pm film thickness). Carrier gas was helium at a head pressure of 95 kPa. The splitless injector temperature was 250°C. The GC oven temperature was programmed from an initial temperature of 55°C to 220°C (20”C/min), then to 245°C (5”C/min). Under these conditions, SBzMA methyl ester had a retention time of about 12 min. The mass spectrometer

195

-

11.7

11.9

12.1

Retention

12.3

time

m/z

91

m/r

201

12.5

(min)

Fig. 1. Selected ion chromatograms for SBzMA methyl ester (m/z 91 and 208) and d7-labelled internal standard (m/z 98 and 215) measured in the urinary extract of a rat treated with NMBzA (5 mg/kg body wt.).

was tuned to monitor ions at m/z 91 and 208 for analyte and 98 and 215 for the deuterated internal standard (Fig. 1). Identification was based on GC retention times and correct surface area ratios for the ions 91/208 and 98/215. The calibration curve for SBzMA was linear from 0.5 to 200 ng with a correlation coefficient of 0.995. The detection limit for the method was about 10 ng SBzMA/0.5 ml rat urine.

50

100

150 Mass/Charge

200

250

300

50

00

150 Mass/Charge

200

250

300

fi&-;,

C 274

50

100

150 Mass/Charge

200

250

:

Fig. 2. Mass spectra of (A) urinary, (B) authentic (C) d,-internal standard SBzMA methyl ester.

.z 0 4,20oa ‘c .E 3

b

300

and

24

16-

Results Identification

of SBzMA

in the urine of rats

treated with NMBzA The presence of SBzMA in the urine was verified by full-scan GC-MS for a rat treated with NMBzA at 5 mg/kg (Fig. Za). Both GC retention time and mass spectrum corresponded to those of authentic SBzMA (Fig. 2b). The mass spectrum of d7-SBzMA is shown in Fig. 2c.

2.5

NMBzA

(mg/kg)

Fig. 3. Dose-dependent urinary excretion of SBzMA by rats administered NMBzA. Each point represents the mean value f S.E. from four animals. In some cases error bars are within the symbols. ( n ) Wistar, ( l ) BD VI, ( A) Fischer 344 and (+) Sprague-Dawley rats. *Statistically different at P < 0.05 (Student’s t-test) as compared to Wistar or Fischer rats.

lected during the first 12 h. At dose levels I 2.5 mg/kg, Sprague-Dawley and BD VI rats excreted higher levels of SBzMA than Wistar or Fischer 344 rats, while at 5.0 mg/kg, the 24 h urine of Wistar rats contained the highest level of conjugate, the quantity excreted by the latter rat strain being 3-fold that for BD VI rats. The same ratio applied for all collection times investigated (Fig. 4). v)

I

op

6

0

12

24

18

Time After Administration

Contribution of N-demethylation versus Ndebenzylation to the formation of SBzMA excreted in the urine of rats treated with NMBzA Benzaldehyde, an N-debenzylation product of NMBzA metabolism has been shown to form SBzMA in rats [7]. When administered intragastrically to Sprague-Dawley rats in amounts corresponding to that derived from 100% metabolism of 0.017 or 0.034 mmol/kg NMBzA through N-debenzylation, the quantities of SBzMA excreted in the 24-h urine represented only 12 and 30%) respectively, of those in the urine of rats treated with NMBzA (Table 1).

(h)

Time course of urinary excretion of SBzMA Fig. 4. (fig/rat) from rats administered NMBzA (5 mg/kg body wt.). The quantities of SBzMA for 6- 12 h intervals are plotted. Each point represents mean value f S.E. from four animals. ( m) Wistar, ( l ) BD VI and ( A ) Fischer 344 rats.

Excretion of SBzMA in rat urine as a function of dose of NMBzA, time after administration and species The amount of urinary SBzMA increased with the dose of NMBzA (1.5-5 mg/kg) administered intragastrically to Wistar, Fischer 344 and Sprague-Dawley rats (Fig. 3). For BD VI rats the level of excretion reached a plateau at 2.5 mg/kg (Fig. 3). The kinetics of SBzMA excretion by rats treated with NMBzA (5 mg/kg body wt.) was studied in Wistar, Fischer 344 and BD VI rats. Most of the SBzMA was excreted within 24 h for the three rat strains investigated (Fig. 4), the highest amounts in the urine being col-

Table 1. Urinary excretion

of SBzMA following

Numerous studies on the metabolism of NMBzA in vitro have shown that this asymmetric nitrosamine can undergo two types of a-hydroxylation to generate either a methylating species and benzaldehyde or a benzylating and species formaldehyde [4,5,8,16]. Our previous studies have indicated a glutathione-dependent reduction of

treatment

Urinary SBzMA (nmo1/24

Dose

Discussion

of Sprague-Dawley

rats with NMBzA or benzaldehyde.

h) from rats administered: -

(mmoi/kg) Benzaldehyde

(A)

NMBzA (B)

A B

(9%) 5.1 f 0.8 (4)” 21.5 =t 3.5 (4)

0.017 0.034 “Mean f

S.E. (number

of rats).

41.4 72.9

f 2.3 (4) + 4.1 (4)

12 30

x 100

197

mutagenicity in S. typhimurium by benzylating species derived from NMBzA in the presence of liver preparations from various rat strains [lo]. These observations encouraged us to search for urinary SBzMA in the rats treated with NMBzA. As indicated in this report, the mercapturic acid present in urinary extracts was clearly identified as SBzMA (S-benzyl-N-acetylcysteine) on the basis of co-chromatography with authentic compound and comparison of mass spectra. Urine of untreated rats also contained low quantities (up to 1.4 pg/24 h urine) of SBzMA. This indicates the presence in rat diet of substances, yet to be characterized, which can be metabolized into benzylating species. Hydroxylation of the methylene group of NMBzA leads to the formation of methylating species and benzaldehyde, which can be converted into electrophilic species through enzymatic reduction to benzyl alcohol and sulfate conjugation [8]. Furthermore, benzyl alcohol, derived from the reaction of benzylating species with water, may also contribute to the formation of sulfate conjugates. However, since urinary levels of SBzMA following administration of an equimolar dose of benzaldehyde, instead of NMBzA, represented only 12-30% of urinary levels for the nitrosamine-treated rats, SBzMA in the urine of NMBzA-treated rats would appear to derive mainly from benzylating species resulting from the hydroxylation of the methyl group of NMBzA. The prevalence of the latter pathway in the formation of SBzMA may be attributable to a higher rate of methyl oxidation and/or higher rate of GSH conjugation from benzyldiazohydroxide as compared to benzylsulfate and/or may be due to the formation of (i) a glycine conjugate (hippuric acid) from benzoic acid after oxidation of benzaldehyde and (ii) a glucuronide from benzyl alcohol [l]. In a previous study, we showed that with liver S9 from Wistar rats, the mutagenicity of NMBzA was completely suppressed by the addition of GSH, while the reducing effect of GSH was less pronounced in the presence of

liver S9 from BD VI or Fischer 344 rats [lo]. Based on urinary excretion of SBzMA, such rat strain specificity holds true only when rats were administered 5 mg/kg NMBzA. The opposite results obtained at doses I 2.5 mg/kg suggest that BD VI and Wistar rats, while showing similar liver P-450-dependent benzylating capacity [lo], differ in both their content of glutathione transferase(s) and their affinity for benzylating species. This emphasizes the fact that the amount of urinary SBzMA results from the combination of the exposure dose and enzymatic characteristics, i.e. specificity and activity of the P-450/glutathione transferase system. NMBzA has been reported to occur in foods and in the gastric juice of study subjects living in a high-risk area for esophageal cancer [9,11,12]. To assess human exposure to this organ-specific carcinogen, selective, sensitive and non-invasive methods for biological monitoring are required. Urinary excretion of mercapturic acids has been considered to be a useful indicator of exposure to electrophilic agents [3] and specific mercapturic acids have been measured in the urine as an index of human exposure to certain chemicals in the work environment [ 14,151. Although the results of the present study are promising, the detection limits of the present method (10 ng/0.5 ml rat urine) do not allow quantification of this conjugate in the urine of humans exposed to dietary levels of NMBzA. Work is in progress to improve the sensitivity of the method using specific antibodies for the clean-up procedure and negative ion chemical ionization mass spectrometry. It would be of interest to find out whether human urine contains similar background levels of SBzMA and if so, what the dietary sources are. Acknowledgements

The work reported was undertaken during the tenure of a special training award from IARC to Dr D.-X. Lin. The authors are grateful to Dr H. Ohshima for helpful suggestions and to L. Garren for technical assistance. Thanks

198

are also due to Dr J. Cheney for editorial help and to E. Bayle for typing the manuscript.

9

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