Kinetics of metabolism of propene and covalent binding to macromolecules in the mouse

TOXICOLOGY

AND APPLIED

PHARMACOLOGY

73, 363-372 (1984)

Kinetics of Metabolism of Propene and Covalent Binding to Macromolecules in the Mouse KETTIL Department of Radiobiology,

SVENSSON’

AND SW OSTERMAN-GOLKAR

Wallenberg Laboratory,

University of Stockholm. S-106 91 Stockholm, Sweden

Received April 30. 1983; accepted October 24, 1983 Kinetics of Metabolism of Propene and Covalent Binding to Macromolecules in the Mouse. K. AND OSTERMAN-GOLRAR, S., (1984). Toxicol. Appl. Pharmacol. 73, 363-372. The rate of uptake of propene from air was studied by exposing CBA mice to various wneentrations of the gas in a closed, recirculating all-glass chamber. The rate curves showed a saturable dependence on the propene concentration. The inhalational K, and V,, were calculated to be 800 + 60 ppm and 8 * 0.5 mg (kg body wt)-’ hr-‘, respectively, from a Lineweaver-Burk plot of the rate data. The homologous compound ethene is known to be metabolized in the mouse to ethene oxide. When a trace amount of ‘4c-labeled ethene was administered in combination with a high concentration of propene, the uptake of [‘%]ethene was lower than in the absence of propene, suggesting a competitive interaction in their metabolic pathways. One group of animals were exposed at 20,000 ppm of propene 4 hr/day during 8 consecutive days. Hemoglobin was isolated from the treated group and a control group. AtIer hydrolysis of the protein, two diastereomers of W-(2-hydroxypropyl)histidine were identified in the hydrolysate from treated animals, suggesting that propene, analogous to ethene, is metabolized to the wrres~nding epoxide and showing that the oxidation is not stereospecitic. 2-Hydroxypropylated products were found in hemoglobin from mice treated with “C-labeled propene. The amounts of alkylated products in DNA were below the detection limit. SVENSSON,

Propene is an important industrial compound, used for the synthesis of several three-carbon compounds, such as glycerol, acetone, and isopropanol, and for the production of polymers (Eberson, 1969; Merck Index, 1976). Propene is formed by combustion of organic matter and is present as a contaminant in urban air (Altshuller et al., 197 1; Gordon et al., 1968) and as one of the major olefins in cigarette smoke (U.S. Dept. HEW, 1979). High exposures to propene may occur in connection with its industrial usage. Epoxidation in vivo is a common step in the transformation of many unsaturated hydrocarbons, including simple alkenes such as ethene (Ehrenberg et al., 1977; Segerback, ’ To whom wrrespondence should be addressed. 363

1983) and I-hexadecene (Watabe and Yamada, 1975), to more water-soluble products. The transformation of ethene to ethene oxide was indirectly demonstrated in experiments with mice (Ehrenberg et al., 1977; Segerback, 1983) by a determination of 2hydroxyethylated products of guanine-N-7 of DNA from different organs and of nucleophilic amino acid residues in hemoglobin. A direct comparison with ethene oxide showed that the degree of alkylation obtained in different organs and of different nucleophilic sites were consistent with the hypothesis that ethene oxide is the reactive intermediate. A quantitative comparison based on actual uptake of ethene and yield of alkylated products indicates that ethene oxide is the main metabolite (Segerback, 1983). 0041-008X/84

$3.00

Copyright Q I984 by Academic Pms, Inc. All rigJ~ts of repmduchm in any form raervcd.

364

SVENSSON

AND OSTERMAN-GGLKAR

For an estimation of the genetic risk associated with exposure to chemicals, it is necessary to have information about the dose (dose = time integral of concentration) obtained in vivo of electrophilically reactive compounds/intermediates. Stable reaction products of macromolecules may be used for the dosimetry since there is a direct proportionality between degree of alkylation (or other type of substitution) and dose (Ehrenberg et al., 1974; Osterman-Golkar et al., 1976; Lee, 1978). In experimental animals the degree of alkylation of DNA may be determined as a measure of the dose with radiolabeled chemicals. For an estimation of the in vivo dose of electrophilic compounds in man, samples that are obtained easily and with a minimum of discomfort to the individual are required. Alkylated products of hemoglobin have been used for monitoring in vivo doses of ethene oxide and propene oxide in both animals (Osterman-Golkar, 1975; Osterman-Golkar et al., 1976; Segerbick, 1983; Farmer et al., 1982) and man (Calleman et al., 1978; OstermanGolkar, Farmer et al., unpublished data). The present study includes the determination of the degree of binding to DNA and hemoglobin of the reactive intermediate propene oxide formed after exposure of mice to propene. The kinetics of the metabolism of propene were studied by exposing animals to specified concentrations of the chemical in a closed system and monitoring its rate of depletion from the atmosphere (cf. Andersen et al., 1980; Filser and Bolt, 1979; Hilderbrand and Andersen, 1981; Gargas and Andersen, 1982). METHODS Animals Male CBA mice (Anticimex, Sollentuna, Sweden), 12 weeks old, average weight 3 1 g, were used. They were. fed a standard pellet diet and water without restriction.

beled, 119.8 mCi/mmol; radiochemical purity 99%) were obtained from The Radiochemical Centre, Amersham, England. Unlabeled propene (min. purity 99%) was obtained from AGA Special Gas AB, Lidingo, Sweden. The propene was free from contaminating propene oxide as checked by bubbling the gas through a solution of 4-(p nitrobenzyl)pyridine in 0.3 M acetate buffer, pH 4, which was analyzed according to Walles ( 1980). S-(2-HydroxypropyBcysteine was synthesized from Lcysteine hydrochloride (BDH Chemicals Ltd., Poole, England) and propene oxide (Merck-Schuchardt, Darmstadt, FRG) according to Zilkha and Weinstein (1961). N*-(2-Hydroxypropyl)valine2 was synthesized by incubating I-amino-Zpropanoi (50 mmol, Merck-Schuchardt) and cu-bromoisovaleric acid ( 18 mmol, Fluka AC, Buchs SC, Switzerland) in 5 ml of water for 15 hr at 100°C. The alkylated valine was isolated by ion exchange chromatography (see below). Nr-(2-Hydroxypropyl)histidine and N’-(2-hydroxypropyl)histidine were synthesized by reacting propene oxide (14.8 mmol) with N*-acetylhistidine (8.1 mmol, Sigma, St. Louis, MO.) in 20 ml of water at pH 7.4 for 24 hr at room temperature. After reaction the solution was evaporated to dryness and the product was hydrolyzed in 4 M HCl for 4 hr at 90°C. The alkylated histidines were isolated by ion exchange chromatography (see below). 14CLabeled histidine (The RadiochemicalCentre, 345 mCi/ mmol) was acetylated by reacting 50 &i “C-labeled histidine with 0.5 mmol acetic anhydride in 2.5 ml of acetic acid at 120°C for 30 sec.The radiolabeled N’-(2-hydroxypropyl)histidine, to be used as tracer, was synthesized by the method described for the unlabeled product. N-7-(2-Hydroxypropyl)guanine was synthesized by reacting propene oxide (5.0 mmol) with guanosine 5-monophosphate (0.7 mmol, Sigma) in 5 ml of 0.1 M phosphate buffer, pH 7.0, for 16 hr at room temperature. After reaction the solution was evaporated to dryness and the product was hydrolyzed in 1 M HCl for 1 hr at 100°C. The alkylated guanine was isolated by ion exchange chromatography (see below). Fluorescamine was obtained from F. Hollinan-LaRoche & Co., AC. Diagnostica, Basel, Switzerland. Exposure of Animals Experiment A: Kinetics of uptake of propene. Groups of 15 mice, average weight 3 1 g were exposed in an 11: liter all-glass system to various concentrations of unlabeled propene. The propene was transferred from a gas burette into the treatment chamber. The air in the chamber was circulated by a propeller and expired carbon dioxide was absorbed on Ascarite. The chamber was connected via a water trap to an oxygen supply. The concentration of

Chemicals [‘QPropene (uniformly labeled, 0.49 mCi/mmol; radiochemical purity 99%) and [“Clethene (uniformly la-

2 The nomenclature of N’, N*, and N’ is in accordance with recommendations of IUPAC-IUBS (1972).

METABOLISM

AND BINDING

propene in the air was determined by gas chromatography (see below) and was followed for at least one half-life. A stainless-steel needle, which pierced a membrane in the treatment chamber, was used to transfer the air in the chamber to a lo-ml evacuated blood collecting tube. The initial concentrations of propene were 95, 250, 315, 380, 500,780, 1500, and 17 15 ppm, respectively. All exposures were performed within a period of 10 days. Experiment B: Uptake of [‘*C]ethene in the presence and the absence of unlabeled propene. B 1: I5 mice, average

weight 30 g, were exposed to i4C-labeled ethene for 7 hr in the treatment chamber. The ethene (0.5 &i) was distilled from the ampoule into a 150-ml vessel. Air, 200 ml, was flushed through the vessel with the radiolabeled ethene and transferred to the treatment chamber. The concentrations of “C-labeled ethene (initial concentration: 0.01 ppm) in the air was monitored at intervals by taking 2-ml samples (standard deviation 0.10 ml, defined by taking 25 samples) by a gas syringe and rapidly injecting the gas through a membrane mounted into the cover of scintillation flasks. The flasks were prefilled with scintillation liquid. The inhalation chamber was checked for leakage with a tracer amount of 14C-labeled ethene. The loss of radioactivity during a 24-hr period was estimated to be 5% in the absence of animals. B2: 15 mice, average weight 32 g, were exposed to 14Clabeled ethene (initial concentration: 0.01 ppm, 0.5 &i) together with unlabeled propene (I 260 ppm) for 7 hr. The concentration of propene was determined (cf. Experiment A). The experiment was essentially performed as described under B 1. Experiment C: Acute exposure to [“Clpropene tification of alkylatedproducts in macromolecules.

for iden-

Twelve mice, average weight 26 g, were exposed to 14C-labeled propene for I hr. The propene (350 pCi) was distilled from the ampoule into a 150-ml vessel, containing 2 ml of 1 M H2S04, which was stirred for 10 min (to certify the destruction of propene oxide, a possible, although not likely, radiolytic product of propene). Unlabeled propene, 200 ml, was shaken in a gas burette containing 1 M H,SO, . The propene was then flushed through the vessel with radiolabeled propene and transferred to the treatment chamber. The concentration of i4C-labeled propene3 was measured as in experiment Bl. The mice were killed 13 hr after the treatment. Blood was collected and hemoglobin was isolated as described earlier (Dsterman-Golkar et al., 1976). DNA was isolated from livers, testes,spleens, lungs, and kidneys by the phenol m-cresol 8-hydroxyquinoline method (Kirby et al., 1976).

’ The depletion of propene from the inhalation chamber during the exposure was more rapid than expected, due to an accidental leakage from the system. For this reason the experiment was terminated after 1 hr. Because of the high costs of radiolabeled propene the experiment could not be repeated.

365

OF PROPENE

Experiment D: Chronic exposure to unlabeled propene for determination by chemical methods of alkylated products in hemoglobin. Twelve mice, average weight 25 g,

were exposed by passing a 2% mixture of propene in air through the chamber at a constant flow rate of 30 liters/ hr. The animals were exposed 4 hr/day during 8 consecutive days. Blood was collected immediately after the last exposure and hemoglobin was isolated. Twelve mice of the same strain, sex, and age were used as controls. Gas Chromatographic

Analysis

of Propene

A Varian 3700 gas chromatograph equipped with a flame ionization detector and a 50 X 0.3I-cm stainless steel column packed with 70- to 230-mesh activated ammina was used. Aliquots of 20 81 from the blood collecting tubes were injected on the column (column temperature 60°C; flow rate 30 ml/min). The concentration of propene in the treatment chamber was calculated by comparing the automatically integrated peak area of propene to that of a standard. Determination of Hydroxypropylated moglobin and DNA, Respectively, Measurements (Experiment C)

Products in Heby Radioactivity

Due to the high concentration of ghrtathione in red blood cells, alkylated products of this compound might be a contaminant of the hemoglobin preparation. To sep arate the protein from low molecular weight compounds, 820 mg of hemoglobin was dissolved in 10 ml of 0. I M formic acid (containing urea, 6 M, and dithiothreitol, 50 mM). Glutathione, 10 mg, was added (as a reference for low molecular compounds) and the sample was chromatographed on a Sephadex G-25 column (50 X 2.5 cm) which was eluted with 0.1 M formic acid. The fractions containing the protein were dialyzed for 3 days against 1 mM phosphate buffer, pH 7. After evaporation to dryness, the protein was dissolved in 6 M HCl(1 mg/O. 1 ml) and hydrolyzed in an evacuated tube for 15 hr at 120°C. The hydrolysate was evaporated to dryness. The residue was dissolved in 5 ml of water and incubated for 1 hr at 37°C to hydrolyze the 2-chloropropylated products that may be formed from 2-hydroxypropylated products during the protein hydrolysis (cf. Calleman et ab, 1978). The hydrolysate was divided into two main fractions (400 ml of 1 M HCI and 400 ml of 2 M HCl) on a Dowex 5OW-X4 (26 X 1.2 cm). The 1 M HCI fraction was evaporated, carrier S-(2-hydroxypropyl) (5 mg) was added, and the amino acids were separated on a Dowex 5OW-X4 column (67 X 1.0 cm) eluted with 1 M HCI. Fractions of 3 ml were collected. S-(2-HydroxypropyBcysteine eluted after 180 ml. Fractions 5 1 through 83 were rechromatographed on a longer column (I 12 X 1.O cm) with carrier N*-(2-hydroxypropyl)vahne (25 mg) added. The column was first eluted with 440 ml of 0.5 M HCI, then with 1

366

SVENSSON AND OSTERMAN-GOLKAR

HCI which was collected in 1I-ml fractions. S-(2-Hydroxypropyl)cysteine and N*-(2-hydroxypropyl)vahne eluted after 90 and 120 ml of 1 M HCI, respectively. The 2 M HCI fraction, with the two isomeric hydroxypropylhistidines (5 mg of each) added as carriers, was chromatographed on a Dowex 5OW-X4 column (67 X 1.0 cm) eluted with 2 M HCI. Fractions of 5 ml were collected. The N’-(2-hydroxypropyl)histidine and N’-(Z-hydroxypropyl)histidine eluted after 170 and 2 10 ml of 2 M HCI, respectively. Due to the insufficient resolution, the alkylated histidines were rechromatographed on an Aminex A-5 column (58 X 0.9 cm) connected to a Cheminert CMP-1 pump (Laboratory Data Control). The column was eluted with 50 ml of 0.1 M phosphate buffer, pH 6.0, followed by 0.1 M phosphate buffer pH 7.5. N’-(2-Hydroxypropyl)histidine and N’-(2-hydroxypropyl)histidine eluted after 60 and 75 ml, respectively, of the latter buffer. Finally, the hydroxypropylhistidines were rechromatographed separately on a Dowex 5OW-X4 column (77 X 1.O cm) eluted with 2 M HCl. The amino acids in the fractions were identified by ninhydrin with thin layer chromatography (OstermanGolkar et al., 1976). The degree of alkylation of hemoglobin was determined from the radioactivity in peaks corresponding to alkylated amino acids. DNA from livers, 55 mg, was hydrolyzed in 1 M HCI for 1 hr at 100°C. After addition of N-7-(2-hydroxypropyl)guanine, the hydrolysate was chromatographed on a Dowex 5OW-X4 column (26 X 1.3 cm) with 1.5 M HCl. Fractions of 6.3 ml were collected. The fractions (51 through 59) containing N-7-(2-hydroxypropyl)guanine were pooled and rechromatographed on an Aminex A-5 column (2 I X 0.9 cm) which was eluted with 0.4 M ammonium formate, pH 4.3, at 37°C. The separation was monitored by uv and radioactivity of the fractions was measured. Pooled DNA, 238 mg, from testes, spleens, lungs, and kidneys was treated in the same way. M

Determination moglobin

of N’-(2-Hydroxypropyl)histidine by Means of HPLC (Experiment

in HeD)

Hemoglobin from the exposed mice (0.98 g) and the control mice (1.02 g) was dissolved in 6 M HCI. N’-(2Hydroxypropyl)histidine (8800 dpm; 1.2 X IO-* nmol) was added to each sample as a radiolabeled tracer. The hemoglobin was hydrolyzed and the residue treated with water as described above. The hydrolysates were applied to a Dowex 5OW-X4 column (22 X 1.4 cm) and eluted with 400 ml of 1 M HCI followed by 2 M HCl, which was collected in 13 ml fractions. N’-(2-Hydroxypropyl)histidine eluted in fractions 4 through 10. These fractions were evaporated to dryness and rechromatogmphed on an Aminex A-5 column (54 X 0.9 cm) eluted with 40 ml of 0.1 M phosphate buffer, pH 6.0, followed by 0.1 M phosphate buffer, pH 7.5. The alkylated histidine eluted after 90 ml of the pH 7.5 buffer and was desalted on a Dowex 5OW-X4 column (36 X 0.9 cm) eluted with 160 ml of 1

M HCl followed by 2 M HCI. The fractions containing radioactivity were pooled, evaporated to dryness, and redissolved in 1 ml of distilled water. A method developed by Nakamura and Pisano ( 1976) provides a useful approach to the determination of N’alkylated histidines. After heating in acid, the fluorescamine derivatives of histidines containing an unsubstituted nitrogen in the r-position together with a free NH, in the side chain are intensely fluorescent, while the fluorescence of other fluorescamine-labeled compounds disappears. Samples of IO ~1 from the above discussed I -ml aliquots were transferred to polypropylene test tubes (1 ml), and 100 ~1 of 0.2 M sodium borate buffer, pH 9.0, was added. Fluorescamine reagent, 100 ~1 (20 mg of fluorescamine in 100 ml of acetonitrile) was rapidly added, and the tubes were vigorously shaken. After 5 min at room temperature, 100 ~1 of 2 M HCl was added and the tubes were incubated at 80°C for 1 hr. After the derivatization, N’-(2-hydroxypropyl)histidine was analyzed on a high performance liquid chromatograph (Constametric II, Laboratory Data Control) equipped with a Spherisorb C,a column (25 X 0.46 cm). The column was eluted with a linear gradient of 50 mM acetate buffer, pH 4.5, in MeOH:H20 (38:62) to MeOH:H*O (79:21) at a flow rate of 1.5 ml/min. The fluorescence (excitation at 360 nm, fluorescence 418 to 700 nm) was monitored by a filter fluorimeter (Fluoromonitor III, Laboratory Data Control). The quantitative determination of N’-(2-hydroxypropyl)histidine was made from the recorder trace by measuring the peak-area compared to a standard.

Determination

of Radioactivity

Radioactivity was measured with an Intertechnique SL 30 liquid scintillation spectrometer with external standardization. Protein samples were dissolved in 1 ml of 0.01 M NaOH and counted after addition of 10 ml of Instagel (Packard). Fractions from the ion exchange columns were counted in equal amounts of Instagel. Air samples were measured in toluene containing 5 g PPO and 0.05 g POPOP per liter.

RESULTS

Kinetics of Metabolism

of Propene

To study the metabolism of propene, mice were exposed to initial concentrations of the compound ranging from 95 to 17 15 ppm (Experiment A). The concentration of propene in the treatment chamber was determined at intervals and plotted as a function of exposure time. The rate of uptake, u, 100 to 150 min after onset of exposure (where equilibration

METABOLISM

AND BINDING

OF PROPENE

4.

367

t .

CL

0 0

SC0

K,OXl

c3o.x

PPM

PRCPENE

&

FIG. 1. (A) The rate of uptake of propene, u, (expressed in mg (kg body wt)-’ hr -I, estimated 100 to 150 min after the onset of exposure plotted versus concentration of propene. The smooth curve is the line calculated using the parameters K,,, = 800 ppm and I’,,,, = 8 mg (kg body wt-’ hr-’ (see Fig. 1B). (B) A Lineweaver-Burk plot used to present rate data for propene; u is expressed in mg (kg body wt)-’ hr-’ and the air concentration, [S], in ppm.

between the gas and the blood and richly perfused tissues is assumed to be reached, cf. Andersen et al., 1980 and Gargas and Andersen, 1982), was estimated from the slope of the curve at this time point and expressed in milligrams propene per kilogram body weight per hour. When the observed rates (u) were plotted versus atmospheric concentration of propene, a rectangular hyperbola was obtained (Fig. lA), indicating that, in the range of concentrations studied, the rate-limiting step is a saturable enzymatic process (metabolism) following Michaelis-Menten kinetics. The parameters K,,, (the concentration in the air at which uptake proceeds with half the maximum rate) and V,.,,, (the maximum rate of uptake) were determined to 800 f 60 ppm and 8 f 0.5 mg (kg body wt-’ hr-‘, respectively, with a Lineweaver-Burk representation of the Michaelis-Menten equation (see Fig. 1B).

Experiments by Segerblck ( 1983) have demonstrated that ethene oxide is a main metabolite of ethene in the mouse. It may therefore be expected that the homologous compound propene enters into the same metabolic pathway. When 14C-labeled ethene (0.5 PCi, 0.01 ppm) was administered in combination with a high concentration of propene (1,260 ppm, Experiment B2), the uptake of ethene was lower than in the absence of propene (Experiment Bl), suggesting a competitive interaction in their metabolic pathways (Fig. 2). The rate of uptake of ethene in Experiment Bl was consistent with several earlier experiments (cf. Segerback, 1983). Binding to Macromolecules The following reaction pathway was proposed for propene:

368

SVENSSON

CH,-CH=CHz

AND

OSTERMAN-GOLKAR alkylation of nucleophilic sites (Y-) e.g. in macromolecules

metabolism -CH,-CH-CH2 0’ metabolism by other pathways, I excretion

.CH3-CH-CH2-Y I OH

metabolism, 1 excretion

To demonstrate the transient appearance of propene oxide in tissues, the degree of alkylation of macromolecules was investigated after exposure of mice to radiolabeled propene (Experiment C). Normally, propene oxide is attacked at carbon- 1 in nucleophilic substitution reactions at a pH above 7 (Parker, 1959). Therefore, 2hydroxypropylated products of the amino acids cysteine, histidine, and valine (the N-terminal amino acid) in hemoglobin and 2-hydroxypropylated products of guanine-ZV-7 (normally the most reactive site) in DNA were used as carriers for the separations of radiolabeled products. Ion exchange chromatography of a hemoglobin hydrolysate showed a radioactive peak associated with S-(2-hydroxypropyl)cysteine and N2-(2-hydroxypropyl)valine among the acidic amino acids (see Fig. 3A).

Rechromatography of the fractions containing alkylcysteine and -valine showed that alkylated valine amounts to less than 20% of the total radioactivity in this peak. A major part of the total radioactivity of the hemoglobin (~70%) eluted together with serine, glycine, and alanine and is due to metabolic incorporation of 14C fragments into natural amino acids. The separation of the basic amino acids showed radioactivity associated with 2-hydroxypropylated histidines but also a disturbing background noise of unknown origin. After further purification (see Methods), the radioactivity in the peak of N”-(2-hydroxypropyl)histidine could be determined (Fig. 3B). The degrees of alkylation of cysteine and histidine, calculated from radioactivity measurements, are presented in Table 1. DISCUSSION

FIG. 2. The disappearance of radiolabeled ethene from the inhalation chamber during exposure of mice. The lower and the upper curves show the rates of uptake of ethene in the absence (0) and in the presence (V) of a high concentration of propene (1260 ppm). respectively. Concentration of ethene given in cpm/2 ml of air.

Ion exchange chromatography of DNA hydrolysates did not show any radioactive peak associated with N-7-(2-hydroxypropyl)guanine. By assuming a similar reactivity of propene oxide and ethene oxide (cf. Ehrenberg and Hussain, 198 1, p. 54-55) toward DNA and hemoglobin (for ethene oxide: k nNA(oua+,) = 1 X 10e4 liter (g DNA)-’ hr-’ (Osterman-Golkar et al., 1976) and *HisN’) = 0.3 X 10e4 liter (g Hb)-’ hr-’ (Segerback, 1983)), and by assuming further the same dose of propene oxide in red cells and in the compartments of DNA, approximately the ratio 1:0.3 is expected between the degrees of alkylation of guanine-N-7 of DNA and histidine-N” of hemoglobin. The limits of detection in the present experiment were estimated to 1 nmol (g DNA)-’ and 0.2 nmol (g

METABOLISM

AND BINDING

A

369

OF PROPENE CPM

CPM

120,i 80

I!!

!

ii 40’

k c . ” 27-29

I 45-47

63-85

m-83

’ 15-18

25-26

FRACTION Wo

FRACTION

35-36 Iyo

FIG. 3. Ion exchange separation of amino acids from 820 mg of hemoglobin (cf. Methods Experiment C). (A) Separation of acidic and neutral amino acids on Dowex SOW-X4. Counting efficiency, 70%; background level, 46 cpm. The following amino acids are indicated: 1, se&e; 2, glycine; 3, alanine; 4, S-(2-hydroxypropyl)cysteine (iV2-(2-hydroxypropyl)valine, not added as carrier at this step, eluted simultaneously). (B) N--(2-hydroxypropyl)histidine, rechromatographed on Dowex 5OW-X4. Counting efficiency, 50?&;background level, 12 cpm. The arrows indicate the relative amount of alkylated histidine in the fractions.

DNA)-’ for DNA from livers and pooled organs, respectively. The degree of alkylation of DNA is thus lower than the expected 3 nmol (g DNA)-’ (cf. Table 1). Possible explanations to this discrepancy are (a) chemical instability or repair of the alkyl-

ated product of guanine-ZV-7 during the period ( 13 hr) between exposure and termination and/or (b) a higher dose in red cells than in compartments of DNA. To verify the identity of the alkylation products by chemical determination, mice

TABLE DEGREE

OF ALKYLATION Average

Experiment

OF CYSTEINE air

concentration of propene, exposure time

AND HISTIDINE

1

IN HEMCZCLOBIN

AFTER

EXPOSURE

OF MICE

TO PROPENE

Degree of alkylation of cysteine and histidine in hemoglobin (nmol product (g Hb-‘) CysteineO

Histidine-N’

2

0.9

Histidine-N’

Method of analysis

C

2,830 ppm, lhr

D

20,000 ppm, 8 days X 4 hr/day

70

HPLC: tluorescence

control

12*

HPLC: fluorescence

Ion exchange chromatography: radioactivity determination

’ Includes J&(2-hydroxypropyl)valine (cf. RESULTS). * Background noise interfered at the position for the expected peak. The value should therefore be considered as the upper limit for N’-(2-hydroxypropyl)histidine in the control sample.

370

SVENSSON AND OSTERMAN-GOLKAR

were exposed repeatedly at a high concentration of propene (Experiment D). N’-(2-Hydroxypropyl)histidine was determined by application of the method developed by Nakamura and Pisano (1976) to HPLC. Figure 4 shows the chromatograms of samples isolated from exposed and control animals, the reference derivative of N’-(Zhydroxypropyl)histidine and a mixture of this reference and the sample from exposed animals. These chromatograms clearly show that W-(2-hydroxypropyl)histidine is formed in the hemoglobin of exposed mice. Reaction of synthetic propene oxide (DL) with L-histidine gives two diastereomers (Fig. 4C). The same two products are obtained in vivo (Fig. 4A) which shows that the oxidation

of propene is not stereospecific but yields a racemic mixture. By assuming tentatively that propene oxide is the sole primary metabolite of propene, the rate of formation of the oxide at the air concentration 20,000 ppm may be estimated to be I 1 mg (kg body wt-’ hr-‘. The degree of alkylation of histidine-N’ per unit amount of propene oxide formed may be calculated (see Table 2) and directly compared with data on the degree of alkylation obtained after exposure of animals (rats) to propene oxide (data from Farmer et al., 1982). The rate of detoxification of propene oxide in the two species is not necessarily the same. The comparison indicates, however, that propene oxide is an important reactive metabolite of propene.

FIG. 4. Determination of W-(2-hydroxypropyl)histidine by HPLC afkr derivatization with fluorescamine (cf. Methods Experiment B). The arrows indicate the positions of the two dktereomers of W-(2-hydroxypropyl)histidine. (A) Sample from exposed mice (20 4 injected). (B) Sample from control mice (20 ~1 injected). (C) Standard of W-(2-hydroxypropyl)histidine (20 ~1 injected). (D) Mixture of A) (10 4) and C) (10 PO.

METABOLISM

AND BINDING

371

OF PROPENE

TABLE 2 COMPARISONBETWEENTHE DEGREESOF ALKYLATION OF HISTIDIINE-N’ IN HEMOGLOBIN PER UNIT AMOUNT OF OXIDE FORMED OR ABSORBED, RESPECTIVELY,AITER EXPOSURE OF RODENTS TO PROPENE(MICE) AND PROPENEOXIDE (RATS)

PROPENE

Average air concentration

Experiment D, propene propene oxide (PO)

wt-’

~O,ooOwm 1,300 ppm”

Degree of alkylation of histidine-N’ in hemoglobin (= A) (nmol product (g H@-’ b-1)

hi’) 11 llOb

A-

u

2.2

0.20

12.3”

0.11

’ Data from Farmer et al. (1982). b Calculated assuming a 100% uptake of propene oxide from air: uiMo m r.o = (1.3 ml liter-’ X 0.6 liters min-’ kg-’ X 60 min hr-’ X M,)/24.6 ml mmol-‘, where 0.6 liters min -’ kg-’ is the alveloar ventilation in the rat (Lumb, 1963).

This work strongly suggests that propene is converted in vivo to propene oxide, a known mutagenic (Wade et al., 1978) and carcinogenic (Walpole, 1957; Dunkelberg, 1979) compound. ACKNOWLEDGMENTS This work was financially supported by the Swedish Natural Science Research Council, the National Swedish Environment Protection Board/the Product Control Board, and the Swedish Work Environment Fund. The HPLC procedure was worked out within a project sup ported by U.S. Environmental Protection Agency Grant R 804621-02 during Siv Osterman-Golkar’s visit at the Department of Preventive Medicine and Community Health, University of Texas, Galveston, Texas.

REFERENCES ALTSHULLER, A. P., LONNEMAN, W. A., SUTTERFIELD, F. D., AND KOPCZYNSKI, S. L. (1971). Hydrocarbon composition of the atmosphere of the Los Angeles basin-1967. Environ. Sci. Technol. 5, 1009-1016. ANDERSEN, M. E., GARGAS, M. L., JONES, A. R., AND JENKINS,L. J., JR. (1980). Determination of the kinetic constants for metabolism of inhaled toxicants in vivo using gas uptake measurements. Toxicol. Appl. Pharmacol. 54, 100-l 16. CALLEMAN, C. J., EHRENBERG,L., JANSSON,B., OSTERMAN-G• S., SECERB~CK,D., SVENSSON,K., AND WACHTMEISTER, C. A. (1978). Monitoring and risk assessment by means of alkyl groups in hemoglobin in persons occupationally exposed to ethylene oxide. .Z Environ.

Pathol.

Toxicol.

2, 427-444.

DUNKELBERG, H. (1979). On the oncogenic activity of ethylene oxide and propylene oxide in mice. &it. J. Cancer

39.588-589.

EBERSON, L. (1969). Organiska fdreningar ur de liigre olefinema. In Organisk Kemi, pp. 5 17-522. Almqvist & W&sells, Stockholm. EHRENBERG, L., HIESCHE, K. D., OSTERMAN-GOLKAR, S., AND WENNBERG, I. (1974). Evaluation of genetic risks of alkylating agents: Tissue doses in the mouse from air contaminated with ethylene oxide. Mutat. Res. 24, 83-103. EHRENBERG,L., AND HUSSAIN,S. (198 1). Genetic toxicity of some important epoxides. M&al. Res. 86, l-l 13. EHRENBERG, L., OSTERMAN-GOLKAR, S., SEGERBACK, D., SVENSSON,K., AND CALLEMAN, C. J. (1977). Evaluation of genetic risks of alkylating agents. III. Alkylation of haemoglobin after metabolic conversion of ethene to ethene oxide in vivo. Mutat. Res. 45, 175-184. FARMER, P. B., GORF, S. M., AND BAILEY, E. (1982). Determination of hydroxypropylhistidine in haemoglobin as a measure of exposure to propylene oxide using high resolution gas chromatography/mass sp-ectrometty. Biomed. Mass Spectrom. 9, 69-7 1. FI~SER,J. G., AND BOLT, H. M. (1979). Pharmacokinetics of halogenated ethylenes in rats. Arch. Toxicol. 42, 123136. GARGAS, M. L., AND ANDERSEN, M. E. (1982). Metabolism of inhaled brominated hydrocarbons: Validation of gas uptake results by determination of a stable metabolite. Toxicol. Appl. Pharmacol. 66, 55-68. GORDON, R. J., MAYRSOHN, H., AND INGELS, R. M. (1968). C,C, hydrocarbons in the Los Angeles atmosphere. Environ. Sci. Technol. 2, 1117-l 120. HILDERBRAND, R. L., AND ANDERSEN, M. E. (198 1). In vivo kinetic constants for the metabolism of inhaled hydrocarbon toxicants as determined by gas uptake methods. Toxicologist 7, 86-87. IUPAC-IUB (1972). Commission on Biochemical No-

312

SVENSSON

AND

OSTERMAN-GOLKAR

menclature Symbols for Amino-Acid Derivatives and Peptides Recommendations J. Biol. Chem. 247, 977983. KIRBY, K. S., FOX-CARTER, E., AND GUEST, M. (1967). Isolation of deoxyribonucleic acid and ribosomal ribonucleic acid from bacteria. Biochem. J. 104, 258262. LEE, W. R. (1978). Dosimetry of chemical mutagens in eukaqote germ cells. In Chemical Mutagens: Principles and Methods for Their Detection (A. HolIender and F. J. de Serres, eds.), Vol. 5, pp. 177-202. Plenum, New York. LUMB, W. V. (1963). Small Animal Anesthesia, pp. 404 1. Lea & Febiger, Philadelphia. Merck Index (1976). Ninth ed. pp. 1016-1017. NAKAMURA, H., AND PISANO,J. J. (1976). Fluorescamine derivatives of histidine, histamine and certain related imidazoles: Unique fluorescence after heating in acid. Arch. B&hem. Biophys. 177, 334-335. OSTERMAN-GOLKAR, S. (1975). Studies on the reaction kinetics of biologically active electrophilic reagents as a basis for risk estimates. Thesis, University of Stockholm, Sweden. OSTERMAN-GOLKAR, S., EHRENBERG, L., SEGERB.XCK, D., AND HALLSTROM, I. (1976). Evaluation of genetic

risks of alkylating agents. II. Haemoglobin as a dose monitor. Mutat. Res. 34, l-10. PARKER, R. E. (1959). Mechanisms of epoxide reactions. Chem. Rev. 59, 737-799. SEGERB&K, D. (1983). Alkylation of DNA and hemoglobin in the mouse after exposure to ethene and ethene oxide. Chem-Biol. Interact. 45, 139- I5 1. U.S. Department of Health, Education, and Welfare, Public Health Service (1979). Constituents of tobacco smoke. In Smoking and Health, A Report of the Surgeon General, pp. l-l 19. WADE, D. R., AIRY, S. C., AND SINSHEIMER, J. E. (1978). Mutagenicity of aliphatic epoxides. Mutat. Res. 58,2 17223. WALLES, S. (1980). Determination of reaction rate constants for alkylation of 4-(pnitrobenzyl)pyridine by different alkylating agents. Toxicot. Lett. 5, 161. WALPOLE, A. L. ( 1957). Carcinogenic action of alkylating agents. Ann. N. Y. Acad. Sci. 68, 750-761. WATABE, T., AND YAMADA, N. (1975). The biotransformation of I-hexadecene to carcinogenic 1,2epoxyhexadecane by hepatic microsomes. Biochem. Pharmhcol. 24, 1053-54. ZILKHA, A., AND WEINSTEIN,M. ( 196 1). The reaction of epoxides with amino acids having sulthydryl groups. Bull. Res. Count. Israel lOA, 147-148.