(MOCA) in rats - Springer Link

Arch Toxicol (1990) 6 4 : 4 5 1 - 4 5 8

Archives of

Toxicology 9 Springer-Verlag 1990

Quantification of haemoglobin binding of 4,4'-methylenebis(2-chloroaniline)(MOCA) in rats G. Sabbioni and H.-G. Neumann Institut ffir Pharmakologie und Toxikologie, Universit~it Wtirzburg, Versbacherstrasse 9, 8700 Wtirzburg, Federal Republic of Germany Received October II, 1989/Received after revision March 5, 1990/Accepted March 5, 1990

Abstract. 4,4'-Methylenebis(2-chloroaniline) (MOCA) is used as a curing agent in the production of polyurethane. MOCA is carcinogenic in experimental animals. Haemoglobin adducts have been proposed as dosimeters of aromatic amines for biological monitoring. A quantitative method to determine the adduct has now been worked out in female Wistar rats dosed per os with 3.82, 14.2 and 16.2gmol/kg 14C-ring labeled MOCA or 0.25 and 0.50 mmol/kg unlabeled MOCA. MOCA bound in decreasing amounts to DNA, RNA and proteins of the lung, liver and kidney. Fractions of 0.19% and 0.026% of the dose were bound to the blood proteins haemoglobin and albumin, respectively. MOCA released by hydrolysis from haemoglobin was determined by HPLC with electrochemical detection or by GC-MS. Albumin did not form any hydrolysable adducts with MOCA. Key words: Biomonitoring - Aromatic amines - Protein adducts - Haemoglobin - 4,4'-Methylenebis(2-chloroaniline)

Introduction MOCA is widely used as a curing agent in the manufacture of polyurethane elastomers or foams. The production volume in the USA was estimated at 3500 tons in 1972. No production figures are available for the European coun-

Offprint requests to: G. Sabbioni Abbreviations: D C B Z = 3,Y-dichlorobenzidine, D M B Z = 3,3'-dimethylbenzidine, M O C A = 4,4'-methylenebis(2-chloroaniline), TFA = trifluoroacetic acid anhydride, PFPA = pentafluoropropionic acid anhydride, T F A - M O C A = N,N'-ditrifluoroacetyl-MOCA, TFA-DCBZ = N,N'-ditrifluoroacetyl-DCBZ, A c - M O C A = N-acetyl-MOCA, DiAcMOCA = N,N'-diacetyl-MOCA, NCI = negative chemical ionisation, E1 = electron impact ionisation, SIM = single ion monitoring.

tries. A higher incidence of bladder cancer was reported among workers exposed to MOCA (Cartwright 1983). After oral administration of MOCA, mice developed haemangiosarcomas and hepatomas (IARC monographs 1974; Russfield et ai. 1975), rats developed lung, liver, mammary gland and Zymbal gland tumours and haemangiosarcoma (IARC Monographs 1974; Russfield et al. 1975; Stula et al. 1975; Kommineni et al. 1978). Biomonitoring of exposed workers has been accomplished by measuring the concentration of MOCA in urine (Linch et al. 1971; Van Roosmalen and Klein 1979; Henschler 1983; Ducos et al. 1985; Trippel-Schulte et al. 1986; Cocker et al. 1988). Ducos found from 0.5 up to 1600 gg MOCA per litre urine. MOCA is the major compound found in urine of exposed humans. Other metabolites isolated in experiments with rats (Farmer et al. 1981) have not been detected in humans. Biomonitoring of urinary metabolites is not suitable if exposure occurs irregularly, because the results of urine analysis are strongly dependent on the time of sampling. The measured values can correspond to a maximum or minimum of exposure. Analysis of adducts to macromolecules, on the contrary, reflects the exposure over an extended period. Stable haemoglobin or albumin adducts have a life-time of 120 days or a half-life of 30 days, respectively (Ehrenberg et al. 1974; Neumann 1984; Wogan and Gorelick 1985; Farmer et al. 1987). Aromatic amines have been shown to give a specific adduct with haemoglobin, which can be easily hydrolysed in vitro to yield the parent amine, the determination of which has been proposed for use as a dosimeter in biological monitoring. In addition to indicating the internal exposure, the adduct reflects the extent of metabolic activation. Blood protein adducts of carcinogens in humans have been measured, for example, for ethylene oxide (TiSmquist et al. 1986), 4-aminobiphenyl (Bryant et al. 1987) and aflatoxin (Gan et al. 1988). As part of our research on biomonitoring of aromatic amines, binding of MOCA to blood proteins and to DNA of lung, liver and kidney was studied. The main emphasis of this investigation was the development of analytical

452

methods for the quantification of blood protein adducts in rats. The final goal is the application of the procedures for the exposure control of humans in an occupational environment. Materials and methods Ring labeled 14C-MOCA with a specific activity of 58 mCi/mmol was obtained from Amersham, West Germany. According to HPLC analysis purity was >95%. Unlabeled MOCA was recrystallised in hot ethanol. Ac-MOCA was synthesised by reacting MOCA with 1.5 mole equivalent acetic acid anhydride and triethylamine at room temperature. Silica gel purification with hexane/ethyl acetate, 10/1, gave Ac-MOCA in 30% yield. Rf values of MOCA, Ac-MOCA and DiAc-MOCA were 0.65, 0.54 and 0.4, respectively, on silica TLC plates in ethyl acetate. DiAcMOCA was synthesised with an excess, 4 mole equivalents, of acetic acid anhydride and 1 mole equivalent of triethylamine. Identity of the samples was verified by 1H-NMR on a 80 MHz Broker instrument, by GC-MS (DiAc-MOCA) and by LC-MS (MOCA, Ac-MOCA) on a 5988 Hewlett Packard instrument equipped with a HP 59980A particle beam LC/MS interface and a HP 5980 GC. The mass spectrum of MOCA matches the reference spectrum of the NBS Database (HP 59988K). The most important fragments of the three mass spectra are: MOCA: m/e (abundance): M++4,270(6%); M++2,268(36%); M+,266(55%); M+-C1,231(100%); M+-HC1,Cl,195(44%). Ac-MOCA: M++4,312(5%); M++2,310(25%); M§ M +C1,275(12%); M+-Ac,265(24%); M+-Ac,CI,231 (100%) DiAc-MOCA: M++4,354(4%); M++2,352(13%); M*,350(20%); M+-CI,315(53%); M+-Ac,CI,273(49%); M+-2Ac,266(34%); M§ C1,231(100%). GC-MS analyses of TFA-MOCA and of DiAc-MOCA were performed on a HP-1 (12 m • • gm) capillary column. The temperatures of the injector and of the transfer line were set at 260 ~C. The flow rate was at 1.5 ml/min. The samples were injected in the splitless mode with an initial oven temperature of 50 ~C, followed by a temperature ramp to 240~ at 50~ GC-MS analyses of 2-chloro-4-methyl-aniline were performed on a DB 1701 column (9 m • mm • 1 lain) with the injector and the interface temperature set at 180*C and the oven temperature programmed from 50~ (1 min) to 145 ~C at a rate of 50 ~C/rain. For EI, the electron energy was 70 eV, the emission current was 300/aA, and the source temperature was 200 ~C. Conditions for NCI with methane were as follows: source pressure about 0.7 torr, electron energy at 240 eV, source temperature 150 ~C, and emission current 300 ].tA. UV-spectra of MOCA from in vivo samples were recorded on a rapid spectral detector 2140 from Pharmacia. Radioactivity of the organs was measured by combustion in a Packard oxidizer 306. Instagel (Packard) was used to measure the 14C-MOCA content of the aqueous DNA samples. Haemoglobin samples were treated with hydrogen peroxide overnight at 37 ~C or for 3 h at 80 ~C prior to addition of scintillation fluid.

Animal experiments Female Wistar rats (225-255 g) were obtained from the Zentralinstitut ftir Versuchstierkunde (Hannover, FRG). They had free access to feed (Altromin 1324) and water throughout. MOCA dissolved in 400 I.tl ethanol/propan-l,2-diol, 1/4 (vol/vol) was administered by gavage. Three animals were dosed with 16.2, 14.2 and 3.82 mmol/kg radioactive MOCA. Two animals were dosed with 0.25 and two with 0.5 mmol/kg unlabeled MOCA. After 24 h the animals were anesthetised with ether and blood (4-5 ml) was taken by heart puncture with a heparinised syringe.

Purification of blood proteins Isolation of haemoglobin. Freshly drawn, heparinised blood was centrifuged for 5 min at 2000 g. After removal of plasma, red blood cells were washed three times in equal volumes of 0.9% NaCI solution and lysed by the addition of EDTA solution (4 volumes, pH 7.5; 10-4M). The cell debris was removed by centrifugation (5 min, 4000 g). Haemoglobin was precipitated from the supernatant by adding 4 volumes of ethanol drop wise while stirring. The precipitate was resuspended and centrifuged after washing with solvents: ethanol/water 8/2 (vol/vol), ethanol, cthanol/ether 1/3, and ether. Haemoglobin was dried over silica in a dessicator and then stored at -18~ C.

Isolation of albumin. Albumin was purified by applying plasma to a Cibacron blue affinity column equilibrated with 10 mM "IRIS, pH 7.5 (Travis et al. 1976; Skipper 1985). After washing with 10 mM TRIS, pH 7.5 and 200 mM KC1 +50 mM TRIS (0.2 M, pH 7.4), albumin was eluted with 200 mM NaSCN +50 mM TRIS pH 7.5. All water solutions contained NaN3 (10 raM). Purity was checked by SDS-PAGE electr0phoresis (11% acrylamide) with Coomassie blue staining. To test if all radioactivity was covalently bound to albumin, the protein solutions were dialysed against phosphate buffer and precipitated in organic solvents. To test for loss of MOCA metabolites during dialysis, an albumin solution was applied to a microconcentrator (Amicon, Millipore, molecular weight cut off at 30000 kDalton). Concentration by centrifugati0n (30 min, 3000 g) was followed by two washings with 0.5 ml phosphate buffer. No radioactivity could be found in the filtrate. Ethanol precipitation of the purified albumin and washing with several portions of cold ethanol did not release any radioactivity.

Purification of DNA, RNA and proteins of lung, liver and kidney. The procedure of Gaugler and Neumann (1981) was followed. Purified liver DNA was further treated with Pronase and extracted with phenol, following a procedure of Maniatis et al. (1982).

Hydrolysis of haemoglobin and albumin Hydrolysis of haemoglobin and extraction of MOCA. Haemoglobin 40 mg was hydrolysed in 3 ml 1 N NaOH and 0.5% SDS for 1 h in the presence of the recovery standard DMBZ or DCBZ. The water phase was extracted twice with 3 ml ether. The combined organic extracts were evaporated to dryness on a speed vac (15 min ca 20 Torr). The residue was reconstituted in 50% MeOH (500 lal) and injected (5 I.tl) for HPLC analysis. Recoveries of MOCA (20 ng, 200 ng), Ac-MOCA (20 ng, 200 ng), DCBZ (20 ng, 200 ng) and DMBZ (200 ng, 1 p.g) from spiked haemoglobin (40 rag) were 84 • 80• 75 • and 58 • respectively, with this method. The identity of the extracted material was verified by GC-MS and UV. TFA (50 lal) and triethylamine (2 gl) were added to the dried (magnesium sulphate) ether extracts of one hydrolysate. After 1 h at room temperature the reaction mixture was evaporated to dryness in the speed vac. The residue was reconstituted in 30 lal ethyl acetate and analysed by GC-MS in the full scan mode. A UV spectrum with a photodiode array detector was obtained after HPLC analysis of six combined hydrolyses (extracts from animals dosed with 0.5 mmol/kg; ca 2 lag MOCA). Covalent binding of MOCA was assured by extracting haemoglobin solutions at pH 7.4. Neutral haem0globin solutions of treated rats were applied to a C-18 Sep-Pak cartridge (Waters) and washed with 5 ml water. Unbound MOCA and the added recovery standard were eluted with 2 ml of methanol. An aliquot of the eluate was analysed by HPLC. It contained 50 times less MOCA than after 9 alkaline hydrolysis of the same amount of haemoglobin. The recovery of MOCA and DCBZ, 9 6 • and 8 0 • respectively, by this work-up procedure was determined in a separate experiment by spiking haemoglobin (40 mg) with 1 lag of each compound (extraction efficiency of MOCA, Ac-MOCA and DCBZ with ether is very poor, 15-20% at neutral pH).

453 Table 1. Binding of 14C-MOCA to haemoglobin, serum albumin, DNA, RNA and protein of liver, lung and kidneys Blood

Liver

Da

Hb HBI b

albumin ABF

1

18

18

DNA CBI d 65

Lung RNA RBI e

Kidney

Prot PBI f

DNA CB!

RNA RBI

Prot. PBI

DNA CBI

RNA RBI

46

86

1009

442

257

65

49

Prot PBI 71

4.3

7

16

73

54

107

(25)g

(319)

211

(39)

(25)

42

3.8

13

18

150

77

160

(29)

(296)

405

(45)

(61)

101

a Dose [mg MOCA/kg body weight] b HBI: [mmol (chemical bound)/tool HB] _- Haemoglobin binding index [mmol(chemical applied)/kg] c ABI: [retool (chemical bound)/mol Alb]

.= albumin binding index

[retool(chemical applied)/kg] d CBI: [/.tmol chemical bound/tool DNA] [retool(chemical applied)/kg] 1 tool DNA = 309 g (Lutz 1979)

= (dpm/mg DNA) 3.09 x 108 dpm/kg body weight

RBI: [~tmol chemical bound/mol RNA] [mmol (chemical applied)/kg] 1 tool RNA = 325 g

= (dpm/mg RNA) 3.25 x 108 dpm/kg body weight

f PBI: [gmol chemical bound/tool amino acids]= (dpm/mg amino acid) x 1.11 x 10s [mmol (chemical applied)/kg] 1 tool amino acids = 111 g

dpm/kg body weight

According to the ratios of the UV absorbances, E26o,/E230and E260/E280, these preparations were not purified to the same degree as the other samples

Hydrolysis of haemoglobin and extraction of 2-chloro-4-methylaniline. In the search for the potential cleavage product 2-chloro-4-methylaniline 40 mg haemoglobin from MOCA-treated animals was hydrolysed in (3 ml) 1 N NaOH and 0.5% SDS for 1 h in the presence of a recovery standard (1 ~g 5-chloro-o-toluidine). The water phase was extracted twice with 3 ml hexane by stirring slowly for 10 min. Possible interphases were reduced by centrifugation. The combined organic extracts were concentrated to 1 ml under a stream of nitrogen. For GC analysis 1 lag of internal standard (2,4,5-trimethylaniline)was added. Recovery (82 + 5%) of 2-chloro-4-methylaniline was determined in a separate experiment by spiking haemoglobin with 1 p.g amine and following the same extraction procedure.

scribed previously. TFA (40 t.tl) and triethylamine (3 I.tl) were added to the dried (MgSO4) ether phases. After 1 h the reaction mixture was evaporated to dryness in the speed vac. The residue was reconstituted in 1 ml ethyl acetate, and 2 lal were analysed by GC-MS with E1 and SIM (Fig. 4). The obtained peak integrals were calibrated against standard solutions (n = 3) of 500 ng DCBZ and 400 ng MOCA derivatized with TFA in ether as described above.

Hydrolysis of albumin. Hydrolysis of albumin was performed as described for haemoglobin. Negligible amounts of radioactivity were extracted.

Quantification. All HPLC analyses were run on a LiChrospher 100

Pronase digestion of albumin. Albumin 40 mg was digested with 4 mg

RP-18 (5 p.m) (125 • ram, Merck) column. HPLC analyses were performed isocratically with a flow rate of 0.8 ml/min with 60% methanol in sodium phosphate buffer (0.02 M, pH 7.2), when DMBZ was used as recovery standard (Fig. 3). MOCA, Ac-MOCA, and DMBZ elute at 8.68, 5.71 and 2.97 rain, respectively. With DCBZ as recovery standard, HPLC analyses were performed isoeratically with a flow of 0.95 ml/min with 53% methanol in sodium-phosphate buffer (0.02 M, pH 7.2). MOCA, Ac-MOCA, DCBZ and DMBZ elute after 18.2, 10.9, 15.5 and 4.0 rain, respectively. An electrochemical detector, model 5100A from ESA (Bedford, Massachusetts, USA), was used with the detector voltage set at 0.8 V and the gain at 8 x 10. The peak areas were evaluated using an integration system from Nelson. The system was calibrated before each analysis by injecting a standard solution. The detector response was linear from 0.1 to 10 ng. Extraction and detector response were checked by adding a recovery standard. It was not necessary to correct the integrals of the peaks, since the yield of the added standard was in the given range. The values for MOCA were only corrected for recovery (84_+6%), which was determined in a separate experiment. The detection limit established with standard solutions was 25 pg for MOCA, DMBZ, DCBZ and 50 pg for Ac-MOCA. For GC-MS, 40 mg haemoglobin was hydrolyzed in the presence of the recovery standard DCBZ (500 ng) and extracted with ether as de-

pronase (Calbiochem, 70000 PUK/g) in sodium phosphate buffer (0.1 M, pH 7.4) for 18 h at 37 ~C. The digest was analysed by HPLC on a Partisil ODS-3 column (250 x 4) with a 50 rain methanol ( 4 - 8 0 % ) gradient in phosphate buffer (0.02 M, pH 7.2). One per cent of the radioactivity coeluted with monoacetyl-MOCA and 8% with MOCA. Thirty per cent of the injected radioactivity was collected with the solvent front. The rest of the radioactivity was spread between 20 and 32 min.

Results

Twenty-four hours after oral administration of MOCA the organ containing most radioactivity (% of total dose) w a s in t h e l i v e r (2.1 ___0 . 5 ) ; r a d i o a c t i v i t y w a s a l s o d e t e c t e d in t h e l u n g ( 0 . 2 8 _ + 0 . 1 6 ) , k i d n e y ( 0 . 1 6 + _ 0 . 1 4 ) , i n t e s tine (0.43_0.21), stomach (0.045_0.023), uterus (0.0176+__0.003), spleen (0.022___0.0046) and blood (0.13___0.02). A l l o t h e r o r g a n s c o n t a i n e d l e s s M O C A . M o s t r a d i o a c t i v i t y w a s f o u n d i n u r i n e a n d f a e c e s . T h i s is i n

454 Table 2. Haemoglobin binding indices of animals dosed with 0.00380.5 mmol MOCA per kg body ,weight,An aliquot of 40 mg haemoglobin was hydrolysedtwice per animal Dose [mmol/kg]

HBI

3.82x 10-3

7.2+0.2 a, b

14.2X 10-3

5.7 •

b

16.2• 10-3

2.6+__0.1b

0.25

4.4+0.9 a, c, d

0.50

5.1 +0.6 a, c, d

a b c d

(0.5 mmol/kg). Only a very small amount (8.93 pg per nag Hb) coeluted with Ac-MOCA. The presence of AcMOCA, however, was not confirmed by GC-MS. The HBIs determined by GC-MS with EI (Fig. 4) were within the range of those found by HPLC. The identity of MOCA obtained from in vivo experiments was determined by HPLC cochromatography, by UV (Fig. 2) and GC-MS (Fig. 1). The detection limit of the GC-MS method with different ionisation techniques was tested with standard solutions of TFA-MOCA, TFA-DCBZ and PFPA-MOCA. With EI the major ions of TFA-MOCA and TFA-DCBZ are at 423 and 409, respectively, and 523 for PFPA-MOCA (Figs. 5, 6). The detection limit for MOCA and DCBZ for EI with SIM was 30 pg (signal/noise = 17). With NCI the detection limit for TFA-MOCA could be lowered to 2 pg (signal/noise = 224) (Fig. 7). For the TFA derivatives of MOCA and DCBZ the base peaks are at 422 and 408, respectively (Fig. 5). The abundance of the rn~ lecular ions are only 15% of the base peak. PFPA-MOCA shows one major fragment at 538 (Fig. 6). The response with NCI is 20% larger for PFPA-MOCA than for TFA. MOCA. The stability of the haemoglobin adduct of MOCA in vivo has been studied by Cheever et al. (1988). In rats the haemoglobin adduct has a half-life of 14 days. In a preliminary experiment with six rats we investigated the additivity of single doses and the amount of MOCA found after 36 days. All rats were dosed with 0.25 mmol/kg (body weight) MOCA. Two rats were killed after 24 h. Four rats received the same dose three times at intervals of 48 h. Two rats were killed 24 h after the last dose and two after 36 days. We found 5.3__+0.6 ng after a single dose and 7.3 +--0.4 ng MOCA bound per mg Hb after three consecutive doses. After 36 days only 0.085 ng/mg Hb was found.

Determinedby HPLC with ECD Determinedby HPLC and radioactivity,one animal per dose Determinedby GC-MS with El and SIM Two animals per dose

agreement with experiments described by Farmer et al. (1981) or Tobes et al. (1983). MOCA bound covalently to macromolecules in the three organs analysed (Table 1). Specific binding to DNA, RNA and proteins was highest in lung, and higher in liver than in kidney. MOCA also bound covalently to the blood proteins haemoglobin ( 0 . 1 8 6 + 0 . 0 8 % of total dose) and albumin (0.0261 +-0.0017% of total dose). These values were calculated assuming a content of 0.945 g haemoglobin and 0.098 g albumin per 100 g body weight (Waynforth 1980) (100% binding would correspond to a HBI of 6800 and to an ABI of 66326). The binding index for albumin remains constant for all doses, but there is a spurious value for haemoglobin. Only negligible amounts are hydrolysable from albumin. The hydrolysable fraction (54%) of the haemoglobin-bound metabolites was analysed by HPLC; 80% coeluted with MOCA and 1% with Ac-MOCA. Binding indices were calculated on the basis of recovered MOCA (Table 2). MOCA released by hydrolysis from 40 mg haemoglobin from the rat with the smallest dose (3.82 • 10-3mmol) could be monitored with the electrochemical detector. The measured radioactivity and the peak area corresponded well. Four animals (two for each dose) received 0.25 and 0.5 mmol/kg of unlabeled MOCA. Quantification was performed by HPLC with an electrochemical detector (Fig. 3) and by GC-MS (Fig. 4). The determined HBI for the two doses was 4 . 4 • (0.25 mmol/kg) and 5.1___0.6

14~1-~

=-=

to

TFI-IIII

/

423

2~

IO~d

97

/

c 2

We have identified MOCA as the sole cleavage producl from haemoglobin adducts after oral administration of MOCA to rats. It is interesting to note that with the bifunctional amines benzidine, DCBZ and 3,3'-dimethoxybenzidine the monoacetyl derivatives were also found

III IIIII:

1 2 ~0~

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Discussion

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Fig. 1. EI mass spectrum of the TFA derivativeof MOCA found in vivo

tt ], ) ,. i,.. 30~

I., 350

. . . . . . . . . 4~0

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~g

455

5000! 4500~

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i,v, I

Fig. 2. UV of standard MOCA compared to MOCA isolated in vivo

3500-~ 30013~ 25002000-:. 1500.

nMSZ

STflNDRRSS

.

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=

G.O Time Cmin.)

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Fig. 4. GC-MS with El and S1M (409,423 amu) of the TFA derivative of MOCA found in vivo (0.5 mmol/kg dose). DCBZ was added as internal standard

flOCR

H-nC-IIOCA

J~.A,

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HOCA

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t

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t

i

Fig. 3. HPLC analysis with electrochemical detection on a LiChrospherRPI8 (125x4 ram, 5 ~tm) column with 60% methanol in sodium phosphate buffer, a Mixture of standard: 5 ng DMBZ, Ac-MOCA and MOCA. b Extract of haemoglobin from a rat treated with MOCA (0.5 mmol/kg). DMBZ was used as internal standard

(Neumann 1988), indicating that two types of N-oxidation products are generated. Excluding one spurious value, the HBIs were constant with different doses, which is in agreement with a linear dose-response relationship. The H P L C method and the G C - M S method gave the same results for the quantification of hydrolysable haemoglobin adducts of MOCA. The advantages of the HPLC method are the speed, the low cost, the simplicity and the absence of any derivatisation reaction. With D M B Z as recovery standard, a H P L C run lasts only 10 min. With D C B Z (whose recovery is more like that of MOCA) the analysis time is doubled. GC-MS analysis is more selective and more sensitive but also more laborious and expensive. In particular, GC-MS with NCI is dramatically more sensitive. The analysis of human samples will show which method is more appropriate for routine application. Is biomonitoring feasible by measuring protein adducts of M O C A in blood samples? In an occupational environment we expect repeated or chronic exposure. With the following assumptions (Tannenbaum et al. 1986) one can expect positive findings with a daily intake larger than 750 ng: in humans and rats the same percentage of the dose binds to haemoglobin, the adduct accumulates (60 times a single dose), 10 ml blood are available for analysis, the detection limit is 25 pg. In urine of exposed workers Ducos et al. (1985) found between 0.5 and 1600 gg per liter. Assuming excretion rates comparable to those of rats (30% of the dose), these workers had a daily intake between 1.7 gg and 5.3 mg. Under such circumstances it should be possible to measure haemoglobin adducts in humans.

456

i',,,.

10000-

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8000 G000

l!II : PFPI-IIII]II

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150

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Fig, 6. EI and NCI mass spectra of the TFA derivative of MOCA

250

300

H~ss/Charge

350

400

450

500

457

Ion 4-08 amu 3500

TFR-DCBZ

3000 2500 2000 o

ca~ o c..o rr

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3500"

TFR-fiGCfl

3808" 2580" 2080"

isolated two DNA adducts from rat liver, lung and kidney DNA which corresponded to the adducts found in vitro. The methylene bridge of MOCA had been metabolically activated, yielding two monocyelic derivatives which reacted with adenosine. They were identified using FAB/MS as N-(deoxyadenosin-8-yl)-4-amino-3-chlorobenzyl alcohol and N-(deoxyadenosin-8-yl)-3-chlorotoluene. Adducts of intact MOCA have not been reported. We could not find any adducts of 2-chloro-4-methylaniline to haemoglobin. Thus, the metabolites reacting with haemoglobin and DNA seem not to be the same. The relationship between haemoglobin binding of MOCA and the target (DNA) dose, may then be more complicated, and needs to be analysed. Cheever et al. (1988) showed a relatively uniform ratio of DNA bound and haemoglobin bound MOCA over a period of 29 days after a single dose. The biological half-life for adducted MOCA in rat globin (14.3 days) and liver DNA (11.1 days) were similar. This underlines the possibility of relating the extent of haemoglobin binding of intact MOCA to genotoxicity.

150~" I

6.0

Conclusions

8.0

Time (min.) Fig. 7. GC-MS with NCI and SIM (408, 422 amu) of standard TFAMOCA (2 pg = 4.35 fmol) and TFA-DCBZ(2 pg = 4.49 fmol) Biomonitoring of blood protein adducts of reactive metabolites has concentrated so far on haemoglobin. Most of them seem to bind to a greater extent to haemoglobin than to albumin. Furthermore, haemoglobin persists longer than albumin. Accumulation produces a steady state level of 60 times that observed with a single day's intake for hemoglobin (Ehrenberg et al. 1974) and 29-fold for albumin (Sabbioni et al. 1987). Especially for aromatic amines, the haemoglobin adducts can be easily quantified, since the predominant adduct is readily hydrolysed. The analytical problem is therefore narrowed down to the quantification of the parent amines. The non-hydrolysable adducts of aromatic amines with albumin have only been charactetised for 4-aminobiphenyl (Skipper et al. 1985). For MOCA we have the exceptional case of an aromatic amine that binds only 7 times more to haemoglobin than to albumin. Segerb~ick et al. (1989) reported even a larger fraction bound to albumin (0.5%) than to haemoglobin (0.05%). The identification of albumin adducts, then, deserves further attention and should be investigated with the rat strain used by Segerb~ick et al. (1989). MOCA binds to the nucleic acids of the liver and especially of the lung. This correlates with the formation of tumours in these organs. Most recently, Kugler-Steigmeier (1988, male Sprague-Dawley rats), Cheever et al. (1988, male Sprague-Dawley rats) Segerb~ick et al. (1989, the rat strain is not specified) and Silk et al. (1989, male Wistar rats) also described the binding of MOCA to DNA of lung, liver and kidney. Kugler-Steigmeier et al. (1989) determined CBI values of 2 3 + 7 , 55___20 and 4 for DNA of liver, lung and kidney, respectively. The CBIs in the present paper are higher but the hierarchy of the CBI values for the different organs is the same. Segerback et al. (1989)

Haemoglobin adducts of MOCA in humans should be detectable at least by GC-MS with NCI. Presently, biomonitoting with haemoglobin adducts method should be used solely for exposure control. The current knowledge of tumour formation is too limited to draw conclusions as to the risk associated with such exposures, based only on correlations between haemoglobin adducts and DNA adducts in target tissues of experimental animals. Only extensive epidemiological studies could perhaps provide the information necessary for risk assessment with the tools of protein dosimetry.

Acknowledgements. This research was supported by the Deutsche Forschungsgemeinschaft, the European Community and the Schweizerischer Nationalfonds(Nr.: 83.518.1.87).We thank Dr G. Birner for the determination of the radioactivity in the organs. The skilful technical assistance of Elisabeth R0b-Spiegel and Elke Adamczyk is acknowledged.

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