11
271 Mutation Research, 54 (1978) 271--281 C) Elsevier/North-Holland Biomedical Press DETECTION OF ALKYLATING AGENTS BY THE ANALYSIS OF AMINO ACID RESIDUES IN HEMOGLOBIN AND URINE 1. THE IN VIVO AND IN VITRO EFFECTS OF ETHYL METHANESULFONATE, METHYL METHANESULFONATE, HYCANTHONE METHANESULFONATE, AND NALTREXONE LAN TRUONG, JONATHAN B. WARD Jr. and MARVIN S. LEGATOR Preventive Medicine and Community Health, Division of Environmental Toxicology and Epidemiology, University of Texas Medical Branch, Galveston, Texas 77550 (U.S.A.) (Received 29 May 1978) (Accepted 2 June 1978) Summary The effect of alkylating agents on the amino acid composition of rat and human hemoglobin has been examined. Because the amino acid compositions of these proteins are well established, the changes in the molar ratios of specific amino acids could be monitored in purified hemoglobin samples. Ratios of his- tidine, which is readily attacked by alkylating agents, to proline, which is not, were consistent in hemoglobin purified from untreated blood samples from 14 rats and 25 human subjects. Treatment of hemoglobin in vitro or in vivo with methyl methanesulfonate, ethyl methanesulfonate, or hycanthone methane- sulfonate resulted in a loss of histidine residues relative to proline or phenyl- alanine. The decrease in histidine content increased with treatment dose and time and reached a maximum at about 15% of total histidine. Treatment of rats with methyl methanesulfonate or hycanthone methanesulfonate resulted in increased urinary excretion of methyl histidines. Introduction The group known as alkylating agents encompasses a large number of sub- stances known to be mutagenic and carcinogenic. The most common alkylating agents are alkyl sulfates, alkyl alkanesulfonates, alkyl halides, and bifunctional Abbreviations: EMS, ethyl methanesulfonate; MMS. methyl raethanesulfonate; PBS, phosphate- buffered saline; RBC, red blood cells.

Detection of alkylating agents by the analysis of amino acid residues in hemoglobin and urine 1. The in vivo and in vitro effects of ethyl methanesulfonate, methyl methanesulfonate,

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Page 1: Detection of alkylating agents by the analysis of amino acid residues in hemoglobin and urine 1. The in vivo and in vitro effects of ethyl methanesulfonate, methyl methanesulfonate,

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Mutation Research, 54 (1978) 271--281 C) Elsevier/North-Holland Biomedical Press

DETECTION OF ALKYLATING AGENTS BY THE ANALYSIS OF AMINO ACID RESIDUES IN HEMOGLOBIN AND URINE

1. THE IN VIVO AND IN VITRO EFFECTS OF ETHYL METHANESULFONATE, METHYL METHANESULFONATE, HYCANTHONE METHANESULFONATE, AND NALTREXONE

LAN TRUONG, JONATHAN B. WARD Jr. and MARVIN S. LEGATOR

Preventive Medicine and Community Health, Division of Environmental Toxicology and Epidemiology, University of Texas Medical Branch, Galveston, Texas 77550 (U.S.A.)

(Received 29 May 1978) (Accepted 2 June 1978)

Summary

The effect of alkylating agents on the amino acid composition of rat and human hemoglobin has been examined. Because the amino acid compositions of these proteins are well established, the changes in the molar ratios of specific amino acids could be monitored in purified hemoglobin samples. Ratios of his- tidine, which is readily attacked by alkylating agents, to proline, which is not, were consistent in hemoglobin purified from untreated blood samples from 14 rats and 25 human subjects. Treatment of hemoglobin in vitro or in vivo with methyl methanesulfonate, ethyl methanesulfonate, or hycanthone methane- sulfonate resulted in a loss of histidine residues relative to proline or phenyl- alanine. The decrease in histidine content increased with treatment dose and time and reached a maximum at about 15% of total histidine. Treatment of rats with methyl methanesulfonate or hycanthone methanesulfonate resulted in increased urinary excretion of methyl histidines.

Introduction

The group known as alkylating agents encompasses a large number of sub- stances known to be mutagenic and carcinogenic. The most common alkylating agents are alkyl sulfates, alkyl alkanesulfonates, alkyl halides, and bifunctional

Abbreviations: EMS, ethyl methanesulfonate; MMS. methyl raethanesulfonate; PBS, phosphate- buffered saline; RBC, red blood cells.

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alkylating agents [4]. Alkylating agents constitute, in numbers, the most sig- nificant single group of mutagens [10]. DNA alkylation generally results in depurination, strand-breakage, or cross-linkage leading to miscoding or tem- plate inactivation. Events causing either miscoding or a lethal lesion may initiate extensive repair [6].

Alkylating agents forming substi tution products with nucleic acids can also alkylate a variety of other biological molecules, including proteins. The alkyl- ated groups in proteins are those of relatively high nucleophilicity such as thiol, carboxylate, or amino groups. Where direct alkylation of DNA is difficult or impossible to determine, it is logical to detect alkylating agents by quantita- tively measuring alkylated residues in protein. This concept of testing pre- sumptive carcinogens--mutagens by evaluating alkylation of amino acids in pro- teins (histidine in hemoglobin) was explored by Ehrenberg et al. [3] and Oster- man-Golkar et al. [11,12] using isotopically labelled compounds.

The present s tudy reports on the reduction of histidine residues in hemo- globin hydrolysates after t reatment with known alkylating agents. This tech- nique does not require radiolabelled compounds, and may be suitable for monitoring human subjects as well as animals. Methyl methanesulfonate (MMS), ethyl methanesulfonate (EMS), hycanthone methanesulfonate, and nal- trexone, which is not known to have alkylating activity, were the chemicals selected for this study.

Materials

Animals Male albino rats (Sprague-Dawley; 250--300 g) were used.

Chemicals MMS and EMS were purchased from Eastman Organic Chemicals Co.

Hycanthone was a gift from Dr. Ernest Beuding of Johns Hopkins University, Baltimore, Md. Naltrexone was obtained from the National Institute of Drug Abuse Lot No. QCD 82042. All chemicals except naltrexone were added to phosphate-buffered saline (PBS) pH 7.2 and thoroughly mixed prior to injec- tion. Naltrexone was dissolved in PBS after heating in a water bath at 37°C for 2 h, followed by vigorous shaking for 10 min. Hemoglobin was prepared according to the procedure of Lehmann and Huntsman [8] as described below.

Apparatus An aliquot mixer and a Junior Orbit Shaker (Labline 3520) were used for

gentle constant mixing of blood, and for the removal of lipoproteins. A Beckman 121M amino acid analyzer, was used for quantitative analysis

of urine and a Beckman 119 amino acid analyzer was used for blood studies. Both the Beckman 121M and 119 analyzers employed a single column of Durrum DC-6A resin. The 121M is sensitive to about 1.0 nmole/ml and the 119 to about 2 nmole/ml. A Mettler model H20 balance sensitive to 0.01 mg was used to weigh small quantities of chemicals.

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Methods

Preparation and analysis of hemoglobin hydrolysates

In vitro studies Fresh whole blood from normal subjects was collected in heparinized tubes

and centrifuged at 3000 rpm for 5 min to remove plasma and lymphocytes. 2 ml of red blood cells (RBC) were transferred to 13 ~ 100 mm tubes and

the test chemical added at various concentrations. The contents were mixed in an aliquot mixer and incubated for various time intervals prior to purification.

The purification was carried out as follows. The RBC's were incubated in 3--4 volumes of saline on the mixer for 2 min then allowed to stand for an additional 10 min to shrink the cells after which the saline was removed by cen- trifugation. The procedure was repeated 2--3 times until the supernatant was

1 volume clear. The cells were then lysed in one volume of cold-water. Then ~- carbon tetrachloride was added and the mixture shaken at 320 rpm for 10 min. The suspension was left at room temperature for 10 min then centrifuged at 7000 rpm for 10 min. The supernatant was filtered through two Whatman No. 1 filters. The heme was removed by adding 1 ml of the hemolysate drop- wise with constant stirring to 15 ml of 1.5% HC1 in acetone, at 0°C. The pre- cipitate was removed by centrifugation and washed repeatedly with acetone at 0 ° C, until the supernatant was clear, and finally dried under vacuum. After dry- ing, 0.1 mg of Hb was hydrolyzed in an excess of 6 N HC1 at 110°C for 24 h. These conditions maximally released phenylalanine, proline and histidine. The hydrolysate was dried under vacuum over NaOH to neutralize any remaining HC1. The residue was dissolved in 250--350 pl of buffer and injected into the Beckman 119 amino acid analyzer.

Data derived from the analysis of Hb hydrolysates were evaluated as follows. Using the value obtained for phenylalanine, the known amino acid composition of human Hb [2], and the values for phenylaianine and proline obtained from a series of control analyses (Table 2) a constant was calculated. Multiplication of the values for each amino acid by the constant normalized them on the basis of a standard value for phenylalanine. The purity of the sample was evaluated by comparing the proline value obtained from each analysis to the known value (human Hb) or the standard value derived from the control experiments (rat Hb, Table 2). If the experimental value was within 7% of the expected value the sample was considered adequately pure. The experimental values were within 6% of expected values in the majority of samples. The percentage loss of histidine residues was calculated from the decrease in the normalized amount of histidine as compared to control values.

Animal treatment The chemicals were injected i.p. into rats using 5--8 doses for each drug with

3 animals per dose. All Hb preparations were used on the day of blood collec- tion.

Collection and preparation of hemoglobin 24 h after treatment, the rats were killed by cranial insult followed by car-

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diopuncture with a heparinized syringe; approximately 2 ml of blood was ob- tained by this procedure. The RBC's were washed as above, lysed with cold distilled water, shaken with ½ volume of carbon tetrachloride at 150 rpm for 15 min, and allowed to stand for 15 min at room temperature prior to filtra- tion. The hemolysates were refrigerated for 2--3 days until Hb crystals appeared. The precipitate was washed, dissolved in 6 N HC1 and hydrolyzed 24 h at 110°C.

Collection and analysis of urine samples 3 rats were housed in a single metabolic cage and urine samples were col-

lected in test tubes, kept on crushed ice, at intervals from 2--24 h post-injec- tion. Aliquots of 0.5 ml of urine were pipetted into 12 X 75 mm tubes, adjusted to pH 10.5--11.0 with NaOH and dried under a stream of air. The residue was redissolved in 0.5 ml distilled water and the proteins precipitated by the addition of 2 ml of sulfosalicylic acid in 0.3 N lithium citrate, pH 2.2 containing 80 nmoles/ml norleucine as an internal standard. It was then cen- trifuged at 7000 rpm for 10 min. The supernatant amino acids were determined on a Beckman 121M amino acid analyzer.

The 121M analyzer was programmed to analyze physiological fluids and was capable of resolving 1- and 3-methylhistidine from histidine and from each other. About 20% of normal urinary histidine is methylated, presumably as a consequence of myosin and actin catabolism [1,10].

Results

In vitro studies The analysis of 25 control blood samples (obtained from The University of

Texas Medical Branch Hospital blood bank) is recorded in Table 1. By our criteria 3 of these samples would not be suitable for analysis, due to

a greater than 7% deviation of the proline value from theoretical value. Table 2 illustrates the results with rat hemoglobin samples.

The disappearance of histidine from human Hb in whole blood after treat- ment with MMS and EMS is illustrated in Figs. 1 and 2. Using concentrations of MMS and EMS of 0.65 and 1.3 mg/ml the maximum loss of histidine residues occurred between 150--180 min for MMS and 60--150 min for EMS.

Naltrexone was not active in the in vitro studies. Further testing was carried out with naltrexone, to determine if the inability to react with histidine may be due to the failure to penetrate the RBC membrane. The compound was incu- bated for 2 h at room temperature (25 ° C) with purified hemoglobin. Following this incubation period, subsequent analysis revealed no detectable loss of histi- dine.

In vivo hemoglobin studies In rats, both EMS and MMS showed a dose-related response when loss of

amino acid residues was determined. Fig. 3 records the results observed when the disappearance of histidine and cysteine residues was measured over a dose range of 10--125 mg per kg in rats with MMS. Fig. 4 indicates the results with EMS over a dose range of 50--250 mg per kg.

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T A B L E 1

H I S T I D I N E A N D P R O L I N E V A L U E S IN H E M O G L O B I N O F N O N - T R E A T E D H U M A N S U B J E C T S

E x p e r i m e n t No. Hi s t id ine Pro l ine Er ro r (%)

275

T h e o r e t i c a l 38 .00 28 .00 a 1 37 .78 29 .52 5 .43 2 37 .03 27 .43 2 .04 3 a 3 5 . 6 0 a 36 .32 a 29.71 a

4 35 .84 26 .32 6 .60 5 37.91 28 .88 3 .14

6 a 38 .17 a 24 .26 a 13 .36 a

7 37 .57 26.51 5 .32

8 38 .38 28 .52 1 .86

9 37 .50 26 .68 4 .71

10 38 .48 26 .92 3 .86 11 a 38 .67 a 23 .98 a 15 .11 a

12 3 7 . 0 5 29 .77 6 .32

13 39 .31 29 .24 4 .43 14 37 .78 26 .90 3 .97

15 38 .53 26 .89 6 .75

16 38 .02 30 .10 7 .48 17 38 .84 30 .00 7 .00 18 38 .18 26 .78 4 .36

19 39 .88 28 .34 1.21

20 38 .41 27.97 0 .11 21 38 .25 29 .76 2.71

22 38 .39 27 .38 2.21

23 38 .72 29 .22 4 .37

24 38 .36 29 .45 5 .17 25 40 .16 28.51 1 .82

Mean value and 38 .11 -+ 0 .98 28 .23 -+ 2 .36 d e v i a t i o n

Mean value and 38 .20 -+ 0 .94 28 .23 -+ 1.29 d e v i a t i o n a f t e r the r e m o v a l o f the e x p e r i m e n t s w i t h high d e g r e e o f i m p u r i t y a

a Er ro r ca lcu la ted f r o m ac tua l va lues o f p ro l ine and t heo re t i c a l value o f prol ine .

After a single i.p. injection of hycanthone methanesulfonate in rats, a 5% decrease in histidine residues was found at the lowest concentration used, 25 mg per kg. Fig. 5 records the results obtained over a dose range of 25-- 75 mg per kg with this drug.

Urine studies Urine was analyzed after treating animals with MMS and naltrexone. 1- and

3-methylhistidine were quanti tated directly. Levels of 1- and 3-methylhistidine increased after t reatment with both compounds with concurrent reduction in the level of histidine. Figs. 6 (MMS) and 7 (naltrexone) illustrate the results of this analysis. In the measurements of urine from untreated animals, 20% of the histidine residues are methylated. The loss of histidine from protein and the appearance of methylhistidine in urine were detected with equal sensitivity at

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T A B L E 2

R A T H E M O G L O B I N C O N T R O L

E x p e r i m e n t N o . H i s t i d i n e P r o l i n e E r r o r a (%)

1 4 2 . 5 8 2 0 . 6 5 5 . 9 7 2 a 3 9 . 9 1 a 2 3 . 6 7 a 7 . 7 9 a

3 4 2 . 3 3 2 1 . 4 2 2 . 4 6

4 4 0 . 1 8 2 3 . 2 0 5 . 6 5

5 a 3 9 . 7 4 a 1 8 . 5 3 a 1 5 . 6 2 a

6 a 4 1 . 9 2 a 2 3 . 8 8 a 8 . 7 4 a

7 4 0 . 9 2 2 3 . 0 7 5 . 0 5

8 3 9 . 4 3 2 0 . 6 0 6 . 1 9

9 3 9 . 8 0 2 0 . 7 1 5 . 6 9

1 0 3 9 . 1 5 2 1 . 2 5 3 . 2 3

11 4 0 . 9 9 2 2 . 1 8 1 . 0 0

12 4 2 . 7 8 2 2 . 0 5 0 . 4 1 13 4 3 . 1 8 2 3 . 5 8 7 . 3 8

14 4 3 . 8 1 2 2 . 7 7 3 . 6 9

M e a n v a l u e a n d s t a n d a r d 4 1 . 1 9 -+ 1 . 5 5 2 1 . 9 6 b ± 2 . 0 7 d e v i a t i o n

M e a n v a l u e a n d s t a n d a r d 4 1 . 3 8 ± 1 . 6 3 2 1 . 9 5 + 1 . 1 0 d e v i a t i o n a f t e r r e m o v a l

o f t h e e x p e r i m e n t s w i t h h i g h d e g r e e o f i m p u r i t y a

a E r r o r c a l c u l a t e d f r o m a c t u a l v a l u e s o f p r o l i n e a n d m e a n v a l u e o f p r o l i n e (b) .

I(X~-I

hi Z 6 C

T-- u) 3 :

4C

2 0

r

IO0-

8C

uJ z 6o 0 F- (/) 1 -

4 0

20

i | i i i i i i i

0 30 60 90 IE) 150 180 ~JO 240 270 0 R E A C T I O N T I M E I N M I N U T E S

| I i

60 120 ISO REACTION TIME IN MINUTES

F i g . 1. T i m e c o u r s e o f h i s t i d i n e loss f r o m h u m a n h e m o g l o b i n a f t e r i n v i t r o t r e a t m e n t w i t h m e t h y l m e t h a n e s u l f o n a t e ( 0 . 6 5 m g / m l ) .

F i g . 2 . T i m e c o u r s e o f h i s t i d i n e loss f r o m h u m a n h e m o g l o b i n a f t e r in v i t r o t r e a t m e n t w i t h e t h y l m e t h a n e - s u l f o n a t e ( 1 . 3 m g / m l ) .

Page 7: Detection of alkylating agents by the analysis of amino acid residues in hemoglobin and urine 1. The in vivo and in vitro effects of ethyl methanesulfonate, methyl methanesulfonate,

277

i,l z s o O t- ¢)

5 4C

- - - - - - - - ____&

20

I I I =

0 25 50 75 I00 125

DOSE (mg/kg )

Fig. 3. Disappearance of cys te ine (4) and hist idine (e) in r a t hemo g lo b in . The a lbino rats were in jec ted i.p.

wi th a single dose of m e t h y l m e t h a n e s u l f o n a t e , and sacrif iced 24 h la ter .

10 mg per kg. In the case of naltrexone, t reatment at concentrations between 100--300 mg per kg resulted in increased urine levels of 1- and 3-methylhis- tidine.

1004

80

uJ Z 6 0

I- (n

40 ;¢

20

80

•60 o I- u)

20

I I I I I I I00 200 300 25 5O 75

DOSE (mg/kg) DOSE (mg/kg)

Fig. 4. Loss of his t idine f r o m ra t h e m o g l o b i n a f t e r in vivo t r e a t m e n t w i th e thy l m e t h a n e s u l f o n a t e . H e m o - g lobin o f 3 ra t s was p o o l e d for the analysis of each c o n c e n t r a t i o n . Th e an imals were t r e a t ed w i th e thy l m e t h a n e s u l f o n a t e by a single i.p. in jec t ion a nd sacrif iced 24 h later .

Fig. 5. Loss of his t idine f r o m ra t h e m o g l o b i n a f t e r in vivo t r e a t m e n t w i th h y c a n t h o n e m e t h a n e s u l f o n a t e . The an imals were in jec ted i.p. w i t h a single dose and sacrif iced 24 h later .

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BO O .......... HISTIDINE .... A---- I_N METHYL HISTIDINE

Z~ . . . . 3_N METHYL HISTIDINE :\, 60

'" I '""..... ........ ~....l U.l Z

I • oo ................

ZO" / . L ~ " ' "

/ . ~ I ~ .. . , . , . A

. . 1 " \ ,~ .... \ / \ t

i t 'l \\\

80

,,, 6O Z

40

20

0 HISTIDINE & I_N MeHIS • 3_N MeHIS

CONVULSION FOLLOWED BY DEATH

o .... 2~ s'o is ,6o ,~s 160 260 360 460 DOSE (mg/kg) DOSE (mg/kg)

Fig. 6. Dose- - response curve for the a p p e a r a n c e of 1 N m e t h y l h i s t i d i n e , and me th y lh i s t i d in e as a pe rcen tage of to ta l his t idine in ur ine of fas ted a lb ino ra ts t r ea t ed w i th m e t h y l m e t h a n e s u l f o n a t e (single i.p. in ject ion) , T he ur ine was co l lec ted 75 m i n a f te r t r e a t m e n t , o his t idine; A 1 N me thy lh i s t i d ine ; A 3 N me thy lh i s t i d ine . Er ror bars ind ica te s t anda rd devia t ion .

Fig. 7. A p p e a r a n c e of u r ina ry me thy lh i s t i d ine in rats a f te r t r e a t m e n t w i th na l t r exone . Ur ine of 3 albino rats , in jec ted i.p. by single dose of n a l t r e x o n e , we re poo led and co l lec ted 2 h a f t e r t r e a t m e n t , o, h is t idine; A, 1 N me thy lh i s t i d ine ; L~ 3 N me thy lh i s t i d ine . Er ror bars indica te s t anda rd devia t ion .

Discussion

There is a definite need in the field of Genetic Toxicology to develop a mea- surement for the initial event in the sequence of reactions leading either to mutat ions or neoplasms. Such an assay should be highly sensitive, quantitative, and direct and should be usable in intact animals including man. The pioneering studies of Ehrenberg et al. [3] illustrate the possibility of achieving this goal by the determination of alkylation in macromolecules, such as hemoglobin. The approach taken here did no t look directly for alkylation. Instead, by taking advantage of the fact that the amino acid composit ions of human and rat hemoglobins are well established [2] we looked for changes in the ratios of amino acid residues which react with alkylating agents such as histidine or cysteine to stable amino acids such as proline and phenylalanine. Treatment of hemoglobin in vitro or in vivo with EMS, MMS or hycanthone methanesul- fonate resulted in a decrease in the molar ratio of histidinyl to prolyl residues which was approximately linear with dose. With all t reatments the maximum loss of histidine residues was 15--20%.

The methods developed by Ehrenberg et al. [3] using radioisotopes permit the determination of rate constants for the alkylation of histidine by specific compounds, and the biological half-life of those compounds. From these con- stants the expected degree of alkylation of histidine caused by exposure of an animal to a specific concentrat ion of an agent can be estimated [11]. The

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279

degree of alkylation of histidine in hemoglobin expected after exposure to the concentrations of MMS or EMS used in these experiments would be on the order of 0.1--0.01%. Clearly the disappearance of histidine observed by this technique is several orders of magnitude greater than that predicted from the radioisotope experiments of Ehrenberg et al. [3].

Although the formation of alkylated derivatives of histidine does not account for its disappearance from hemoglobin hydrolysates the dose-respon- sive nature of histidine loss suggests that it results from some interaction of histidinyl residues with the alkylated agents. At this point the mechanism of the response is not known.

These studies indicate that histidine is lost from rat hemoglobin after either in vitro or in vivo exposure to the direct-acting alkylating agents MMS or EMS. A similar decrease in histidine was observed after the in vitro exposure of human hemoglobin indicating that the effect is not specific to rat hemoglobin an that it does not result from a metabolic response of the intact animal. The results obtained in the rat s tudy with MMS indicate that cysteine as well as

• histidine residues are lost after treatment. Histidine is more stable to oxidation than cysteine and therefore is more suitable for this analysis.

The analysis of urine measured 1- and 3-methylhistidine directly and indi- cated an increase in the proport ion of methyl derivative in total urinary histi- dines with increasing exposure to hycanthone methanesulfonate and naltrexone. The relationship of the urinary methylhistidine levels to the dis- appearance of histidine from Hb is not clear. Naturally occurring methylhisti- dines derived from actin and myosin catabolism make up about 20% of urinary histidines. The elevated levels observed after exposure to alkylating agents could result from increased muscle damage as well as from the alkylation of plasma amino acids.

At the present time there is no direct evidence that the antischistosomal agent, hycanthone methanesulfonate, can alkylate macromolecules. Rohrback et al. [13] proposed that this intercalating agent [9,14] might be an alkylating agent [5] after conversion to the reactive ester with the carbonyl produced directly from the hydroxymethy l group [9], see Fig. 8.

Our results indicate that hycanthone methanesulfonate causes the loss of histidine residues from hemoglobin hydrolysates at low concentrations (5% of histidine lost at 25 mg/kg). In urine an increase in methylhistidines is also ob- served at 25 mg/kg.

With the narcotic antagonist, naltrexone, no direct effect on hemoglobin was detected on direct addition to human hemoglobin nor in rat hemoglobin analy- sis. The increased proport ion of methylhistidines observed when the urine of treated animals was analyzed (100--300 mg/kg) in contrast to the negative results observed in the direct hemoglobin analysis could be explained by the failure of the compound or active metaboli te to penetrate the erythrocyte mem- brane. However, the s tudy using purified hemoglobin indicated that even after direct exposure to the protein, naltrexone does not alter the level of histidine residues. The ability of a metaboli te of this narcotic antagonist to interact with histidine residues in purified hemoglobin has ye t to be determined, addi- tionally, the observation with naltrexone could be due to a secondary effect coincidental with the findings of increased alkylated residues in urine.

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280

mutagen reactive ester alkylat ing specie

0 NHR ~ O NHR I O NHR

CH20H CH2- OSO3 CH2~) (O)

hyconthone R= - (CH2)2-N-(CH2CH3) 2

CN;,~ CH 2 et CH 2

+ H ~

(b)

Fig. 8. (a) s h o w s the c o n v e r s i o n o f h y c a n t h o n e to a r eac t ive c a r b o n i u m ion , via t he h y c a n t h o n e s u l f o n a t e es ter . (b) s h o w s the poss ib le r e s o n a n c e f o r m s of h y c a n t h o n e c a r b o n i u m ion .

With the chemicals used in this study, experiments were confined to the analysis o f amino acid composit ion in rats. This analysis, in all l ikelihood can be performed as readily for human subjects as in experimental animals. The base-line data (control samples in the human subjects} would suggest that the consistency encountered in the hemoglobin analysis may be of the same order as in rats.

The simplicity of this method, requiring a minimum of sample processing and the relatively strong effect observed give it enough potential to warrant fur- ther investigations to establish the relationship of histidine loss to alkylating activity.

Acknowledgements

We acknowledge the support of the Environmental Protection Agency Grant No. 4-14562-671135 and National Institute of Drug Abuse Grant No. 6-14546- 671135.

References

1 B r o w n , D .D . , O .L . Silva, P.B. D o n a l d , S.H. S n y d e r a n d M.W. Kies, The m a m m a l i a n m e t a b o l i s m of L-h i s t id ine , J . Biol . C h e m . , 2 3 5 ( 1 9 6 0 ) 1 5 4 - - 1 5 9 .

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