Teratogenesis, Carcinogenesis, and Mutagenesis 3:395-405 (1983)

Dosimetry of Ethylene Oxide in the Rat by Quantitation of Akylated Histidine in Hemoglobin

S. Osterman-Golkar, P.B. Farmer, D. Segerback, E. Bailey, C.J. Calleman, K. Svensson, and L. Ehrenberg Wallenberg Laboratory, Department of Radiobiology, Stockholm University, Stockholm, Sweden (S.0.-G., D.S., C. J.C., K.S., L. E.), and Medical Research Council Toxicology Unit, Medical Research Council Laboratories, Woodmansterne Road, Carshalton, Surrey, England (P. B. F., E . 8 . )

Blood samples were obtained from male Fischer 344 rats exposed to controlled air concentrations of ethylene oxide; 0, 10, 33, and 100 ppm, 6 h/day, 5 days/ week, for 2 years. N-(2-hydroxyethyl)histidine was isolated from hemoglobin hydrolysates and analyzed quantitatively by means of gas chromatography-mass fragmentography and by amino acid analysis. The degrees of alkylation found were 1.3 and 2.8 nmol hydroxyethylhistidine per gram hemoglobin in two groups of unexposed rats, and 14, 34, and 82 nmol per gram hemoglobin, respectively, at the three air levels of ethylene oxide.

Rats of the same breed were given two concentrations of radiolabeled ethylene oxide by IP injection. The degrees of alkylation of amino acids in hemoglobin and of guanine-N-7 in DNA from livers and testes were determined. The degrees of alkylation of liver and testicular DNA were about 150% and 50%, respectively, of the values expected from the degree of alkylation of hemoglobin, basing the expectancy on a direct proportionality between the reactivity of the specific nucleophilic sites and the degree of alkylation obtained at these sites, assuming that the dose of ethylene oxide was the same in the different tissues studied. The in vivo dose of ethylene oxide determined from data on hemoglobin alkylation thus gives a reasonable approximation of the DNA dose. The data were in agreement with a fast elimination of ethylene oxide from tthe tissues, the biological half-life being estimated as about 10 min.

Key words: dosimetry, ethylene oxide, hemoglobin, DNA alkylation

The study performed by S. Osterman-Golkar et a1 reported herein is a separate study from the 2-year ethylene oxide inhalation study performed at the Carnegie-Mellon Institute of Research (CMIR). The sponsors and steering committee for the CMIR study neither participated in nor necessarily support the assumptions andlor conclusions expressed or implied in the study reported herein.

Address reprint requests to S. Osterman-Golkar. PhD, Wallenberg Laboratory, Department of Radio- biology, Stockholm University, s-106 91 Stockholm, Sweden.

0 1983 Alan R. Liss. Inc.

396 Osterrnan-Golkar et al

INTRODUCTION

A long-term toxicity study on ethylene oxide, sponsored by an international group of ethylene oxide producers, has been performed at the Carnegie-Mellon Institute of Research (Bushy Run Research Center), Pittsburg. Blood samples were obtained from male Fischer 344 rats exposed to controlled air concentrations of ethylene oxide (0, 10, 33, and 100 ppm, 6 h/day, 5 daydweek) for 2 years. The degree of alkylation of histidine in hemoglobin was determined as a measure of the in vivo dose of ethylene oxide in the rats. The work was motivated as follows:

1. One of the difficulties in the extrapolation from toxicity data obtained in the Laboratory to risk in man concerns the uncertainty of the in vivo dose obtained of the reactive compound at a given exposure. The degree of alkylation of hemoglobin can be determined as a measure of the in vivo dose of electrophilic compounds in both animals and man. The method makes it possible to circumvent this difficulty.

2. It is not excluded that the exposure to ethylene oxide affects the system responsible for the detoxification of the compound. (There may, for instance, be an induction of epoxide hydrases.) This would lead to a nonlinear relationship between exposure dose and in vivo dose. A determination of the in vivo dose obtained at different exposure levels would therefore be of value for the establishment and interpretation of dose-response relationships.

3. The Carnegie-Mellon experiment offered a unique opportunity to establish relationships between external exposure dose and in vivo dose (determined from the degree of alkylation of hemoglobin and determined from the degree of alkylation of DNA in parallel experiments) and between in vivo dose and response or risk for cancer and heritable damage. Ultimately we wanted to test the hypothesis that a certain degree of alkylation of centers in DNA with a low nucleophilic reactivity gives rise to a tumor incidence that is predictable from the response of the animals to ionizing radiation [cf Ehrenberg, 1979, 19801.

MATERIALS AND METHODS Chemicals

Two supplies of ethylene-14C oxide (22 and 11 mCi/mmole, respectively) were obtained from the Radiochemical Centre, Amersharn, England. Ethylene-d4 oxide was obtained from Merck, Sharp and Dohme, Canada Ltd, Montreal, Canada.

NT-(2-Hydroxyethyl)histidine, unlabeled and labeled with deuterium or 14C, was synthesized by reacting N2-acetylhistidine methyl ester with ethylene oxide in metha- nol-HC1 [Calleman and Wachtmeister, 19791, followed by hydrolysis and ion-ex- change separation of the products. N-7-(2-Hydroxyethyl)guanine was synthesized according to Brookes and Lawley [1961].

Treatments of Animals

Acute exposure (experiment A). Radiolabeled ethylene oxide was dissolved in saline and given to male Fischer 344 rats by IP injection. Two animals received 228 pCi (20.4 pmol) per kg body weight (exp Al) and four animals 61 FCi (2.77 pmol) per kg body weight (exp A2). The animals were sacrificed after 5 h. Blood was collected in heparinized tubes. The red cells were spun down and carefully washed with saline. The globin fraction of hemoglobin was isolated according to Anson and Mirsky [ 19301. DNA

Dosimetry of Ethylene Oxide 397

was isolated from organs essentially as described by Lawley and Thatcher [1970]. The livers and testes were homogenized in 6% p-aminosalicylate (20 ml/g wet weight). The cells were lysed by stirring the homogenate with 1 % sodium lauryl sulphate for 40 min. An equal volume of phenol solution (500 g of phenol, 62 ml of water, 62 ml of m-cresol, and 0.62 g of 8-hydroxyquinoline) was added under vigorous stirring. After 5 min, the extract was centrifuged for 40 min at 8,OOOg. The DNA was precipitated from the water phase by adding 1.5 volumes of -20°C 2-ethoxyethanol and washed with ethanol and ether. The precipitate was dissolved in citrate-buffered saline (15 mM NaCI, 1.5 mM citrate, pH 7.0). When the DNA was dissolved the concentration of the buffer was increased to 150 mM NaCl and 15 mM citrate. Heat-treated RNase, in 0.1 M acetate buffer pH 5.0, was added (10 pglmg of DNA). The RNase treatment continued for 20 min in a shaking bath at 37°C. The DNA was again precipitated with 2-ethoxyethanol and washed with ethanol and ether.

Chronic exposure (experiment B). Exposures of Fischer 344 rats have been performed at the Chemical Hygiene Fellowship Laboratories of Carnegie-Mellon Institute of Research within a program on the toxicity of ethylene oxide. There were three exposure groups (120 males and 120 females per group) that were exposed to 10, 33, or 100 ppm of ethylene oxide for 6 h/day, 5 daydweek. There were also two groups of animals handled in the same way but exposed only to room air. One lyophilized sample of red blood cells was obtained from pools of three or four male rats from each group. These rats had been sacrificed 2 years after onset of exposure. The syringes and cannulae used for sampling of blood were sterilized by y-radiation. The globin fraction of hemoglobin was isolated (see above). The samples were coded

Determination of the Rate Constant for the Reaction of Ethylene Oxide With Histidine-Nr of Hemoglobin

Red blood cells (from 2 ml of blood) from Fischer 344 rats were suspended in 10 ml of 0.1 M phosphate buffer pH 7.4 and treated with radiolabeled ethylene oxide at 37°C (183 pCi/liter). Samples were withdrawn after 20,40, 60, 120, and 180 min and the cells were immediately washed with ice-cold buffer. The globin fraction of hemoglobin was precipitated as described above and 10-mg samples were used for the determination of total radioactivity. Three samples were hydrolyzed and chroma- tographed on a Dowex 50 column (see below) for the determination of the degree of alkylation of specific nucleophilic groups.

Determination of Nr-(2-Hydroxyethy1)Histidine in Hemoglobin and N-7-(2- Hydroxyethy1)Guanine in DNA (Experiment A)

Globin samples (25-50 mg) from animals given radiolabeled ethylene oxide were hydrolyzed in 6 M HCI at 120°C for 15 h. After addition of about 3 mg of carrier N-(2-hydroxyethyl)histidine the hydrolysates were evaporated to dryness, redissolved in 0.1 M HCI, incubated at 37°C for 1 h, and chromatographed on a Dowex 50W-X4 ion-exchange column (1 X 80 cm). The column was eluted with about 300 ml of 1 M HCl followed by 2 M HCl. The amino acids were identified by thin layer chromatography [cf Osterman-Golkar et al, 19761. N-(2-Hydroxyethyl)- histidine was eluted after about 250 ml of 2 M HC1. The degree of alkylation of histidine-NT was calculated from the radioactivity associated with the corresponding fractions.

(NOS. 301-305).

398 Osterman-Golkar et al

Samples of DNA from livers and testes, respectively, were hydrolyzed in 1 M HCI at 100°C for 1 h. After addition of 0.25 mg of carrier N-7-(2-hydroxy- ethy1)guanine the hydrolysates were applied on a Dowex 50W-X4 column (1.5 X 24 cm) eluted with 1.5 M HCI. The peak containing hydroxyethylguanine was resepar- ated on an Aminex A-5 cation exchanger (0.9 X 24 cm). This column was eluted wtih 0.2 M ammonium formate pH 4.7. The flow rate, 24 ml/h, was maintained by a Chromatronic CMP-1 pump. The column temperature was 37°C. The eluate was monitored by a UV detector connected to a recorder. The amount of DNA was calculated from the absorption of the adenine peak obtained from the Dowex column (A,,, at 260.5 nm; 6 = 13.400). The degree of alkylation of guanine-N-7 was calculated from the radioactivity associated with the hydroxyethylguanine peak. Losses were corrected for on the basis of the yield of carrier hydroxyethylguanine.

Isolation, by Ion-Exchange Chromatography, of Nr-(2-Hydroxyethy1)- Histidine of Hemoglobin (Experiment B)

Globin from exposed and control animals (0.5 g/group of animals) was dis- solved in 6 M HC1. Radiolabeled N1-(2-hydroxyethy1)histidine (13,380 dpmhample) was added to serve both as a tracer and as a basis for the calculation of losses during the workup. After addition of a few drops of octanol, the solutions were alternately bubbled with argon and evacuated to a pressure of 5.10-* mm Hg in Pyrex glass tubes. The tubes were sealed under vacuum and the protein was hydrolyzed for 15 h at 120°C. The hydrolysates were then evaporated to dryness, redissolved in 5 ml of 0.1 M HC1, and incubated at 37°C for 1 h to make sure that no N-(2-chloro- ethy1)histidine remained in the hydrolysates [Calleman et al, 19781.

The hydrolysates were then applied to a Dowex 50W-X4 column (1.5 x 27 cm) for a crude separation. The column was eluted with 350 ml of 1 M HCI followed by 2 M HC1. The region of the 2 M HC1 eluate containing the amino acids lysine and histidine was evaporated to dryness and rechromatographed on an Aminex A-5 (Biorad) column (0.9 X 65 cm) which was developed with 0.1 M phosphate buffer pH 7.5. The radioactivity peak of Nr-(2-hydroxyethy1)histidine which eluted after 50 ml was collected and rechromatographed on a Dowex 50 W-X4 (analytical grade) column (1.5 x 85 cm) developed with 2 M HCI. The alkylated histidine eluted after about 700 ml. The appropriate fractions were evaporated to dryness and dissolved in 2 ml of water. This solution was divided into two parts, and the radioactivity of each determined. One fraction was analyzed by mass fragmentography and the other by amino acid analysis.

Quantitative Determination of Nr-(2-Hydroxyethy1)Histidine by Means of Gas Chromatography-Mass Spectrometry (GC-MS)

Derivatization. NT-(2-hydroxy-d4-ethyl)histidine, 0.5 nmole, was added to each sample. The samples were then dried under nitrogen and were esterified by reaction with 200 p1 of 2 M HC1 in methanol for 2 h at 85°C. After cooling the reagents were removed by evaporation under nitrogen at room temperature. Ethyl acetate, 100 pI, and heptafluorobutanoic anhydride, 30 pl, were added and the amino acid esters were acylated for 30 min at 85°C. The reaction mixture was cooled and the solvents removed under nitrogen at 40°C. The product was dissolved in 400 p1 ethyl acetate and partitioned with an equal volume of 0.5 M phosphate buffer (pH 7). The ethyl acetate layer was transferred to a reactivial (1 ml capacity) and then taken to dryness

Dosimetry of Ethylene Oxide 399

under nitrogen at room temperature. The product was redissolved in 40 pl ethyl acetate and aliquots of this solution taken for analysis by GC-MS.

Gas chromatography-mass spectrometry. The mass spectrometer used was a 70-70 VG Micromass double-focusing instrument linked with a VG 2035 Data System and interfaced to a Pye-Unicam Series 204 gas chromatograph. Analyses were made on a 10 m X 0.3 mm Pyrex glass capillary column coated with OV-225 and linked to the mass spectrometer source with glass lined stainless steel tubing (0.4 mm inner diam). Samples were injected into the capillary column using a "falling needle solid sampling device" made from fused silica glass. The inner glass surface of this injector including the glass needle was deactivated with a dilute solution of Carbowax 20 M prior to use.

The chromatographic conditions employed in the analysis were as follows: helium carrier gas flow rate 2 ml/min measured at ambient temperature, injector temperature 200°C and interface temperature 250°C. The column was maintained initially at 100°C for 2 min followed by a 32"Clmin program rise to 240°C. The mass spectrometer operating conditions were: accelerating voltage 4 kV, source temperature 250"C, electron voltage 70 eV, and trap current 200 pamp. Multiple ion detection was carried out by alteration of electrostatic voltage/accelerating voltage; a constant bleed of perfluorokerosene into the source was used to provide a locking mass for the multiple ion detector.

Analyses. The deuterated standard compound and the N-(2-hydroxy- ethy1)histidine were monitored at their (M - COOCH3)' ions at m/e 550 and 546, respectively. Calibration lines were set up using the peak height ratios observed with standard mixtures of these compounds.

Quantitative Determination of Nr-(2-Hydroxyethy1)Histidine on the Amino Acid Analyzer

The samples were evaporated to dryness under nitrogen and were then dissolved in 200 p1 of 0.2 M sodium citrate buffer pH 1.9. Fifty microliters of this solution was used for determination of radioactivity; 100 pI was injected into a Biotronic LC 6OOO E amino acid analyzer equipped with a Durrum DCdA column (25 X 0.6 cm). The column was eluted at a flow rate of 35 ml/h with the following buffers: buffer A (0.060 M sodium citrate, pH 3.15) for 16 min followed by buffer B (0.067 M sodium citrate, 0.100 M sodium chloride, pH 4.25) for 21 min and finally buffer C (0.067 M sodium citrate, 1.40 M sodium chloride, pH 9.65) for 36 min. The temperature program was the following: 51.5"C during the first 18 min, 60.5"C for 77 min, and finally 65.5"C for 15 min. The absorbance was monitored at 570 and 440 nm (ninhydrin detection). The quantitative determination of N-(2-hydroxyethyl)histidine was done by measuring the peak area compared to that of a standard.

Radioactivity Determinations

Radioactivity assays were made using an Intertechnique SL 30 liquid scintilla- tion spectrometer. The counting efficiency was determined by automatic external standardization.

RESULTS AND DISCUSSION

The degree of alkylation of nucleophilic molecules in the cells may be deter- mined as a measure of the dose of an alkylating agendinterrnediate, provided the

400 Osterman-Golkar et a1

target molecule and the product have a sufficient stability [Ehrenberg et al, 1974; Aaron, 1976; Lee, 19761. The degree of alkylation, [RY]/[Y-1, is directly propor- tional to the dose, D (defined as the time integral of the concentration), of the alkylating agent, RX, according to the equation

where ky is the second-order rate constant for reaction of the alkylating agent with the nucleophilic group Y- (RX + Y- + RY + X-).

The second-order rate constant of the reaction in vitro at 37°C of ethylene oxide with rat hemoglobin (kHb) in intact red blood cells was calculated using equation (1) to be 2.3. 1 (g Hb)-' h-I from the initial rate of reaction 780 dpm (mg Hb)-' h-l (see Fig. 1) and the dose, D, assuming that the concentration of ethylene oxide (C, = 406. lo6 dpm/l) remained constant during the initial part of the reaction (ie, D = Coot). Systematic errors involved in radioactivity measurements of globin precipi- tates and of ethylene oxide concentration may result in an uncertainty of f10% in this rate constant. The rate constant for alkylation of histidine-Nr of hemoglobin was determined to be 0.27 k 0.05 - lop4 1 (g Hb)-' h-' after ion-exchange chromatog- raphy of hemoglobin hydrolysates and measurement of the radioactivity associated with the carrier Nr-(2-hydroxyethyl)histidine (Fig. 2).

Table I gives the degree of alkylation obtained in vivo of hemoglobin, of histidine-N. in hemoglobin, and of guanine-N-7 in DNA from livers and testes after treatment of rats with radiolabeled ethylene oxide (exps A1 and A2). Figure 3 illustrates the determination of the degree of alkylation of guanine-N-7 in testes DNA (exp Al).

Knowing the rate constant for alkylation of histidine-Nr in hemoglobin and the degree of alkylation obtained of this nucleophilic center, the dose of ethylene oxide in the red cells may be calculated according to equation (1) ( I kg rat is here approxi- mated to 1 liter). Corresponding calculations, using data on DNA alkylation and the rate constant, 1 * 1 (g DNA)-' h-I (reported values are: 0.9. for calf thymus DNA [Ehrenberg et al, 1974; Segerback, 19831 and 1.0. lop4 for DNA in mouse spleen cells [Ehrenberg et al, 19741, respectively) for alkylation of guanine-N-7 in DNA, show that the dose in liver is somewhat higher (about 50%) and the dose in testes somewhat lower (about 50%) than the dose in erythrocytes (Table I). These results are in agreement with dose determinations in the mouse [Segerback, 19831 where the relative doses to liver DNA, hemoglobin, and testis DNA were found to be 1.3, 1, and 0.6. The studies therefore confirm earlier indications [cf Ehrenberg et al, 19741 that the in vivo dose determined by hemoglobin alkylation gives a good approximation of the dose to DNA.

and 0.96.

The uptake of ethylene oxide from air may be estimated by

0.6 1 kg-' min-' * 60 min h-l - lop6 1 ppm-I 25 1 mol-'

= I .44 x mol kg-' (ppm h)- '

In this estimate the alveolar ventilation (minute volume) of the rats was considered to be 0.6 1 kg-' min-' [Lumb, 19631, and a 100% absorption assumed [Calleman et al, 19781. The injected amounts of ethylene oxide in experiments A1 (20.4. lop6 mol kg-l) and

Dosimetry of Ethylene Oxide 401

0 60 120 180 min

Fig. 1. Radioactivity bound per mg of globin as a function of treatment time. Reaction in vitro between ethylene oxide (C, = 406- lo6 dprn/l) and hemoglobin of intact red cells at 37°C.

150

100

50

I 1 I 1

90 100 110 120 fr.no.

Fig. 2 . Separation of basic amino acids from a hydrolysate of hemoglobin on Dowex 50W-X4. 1) Nr- (2-hydroxyethy1)histidine; 2) lysine; 3) Nr-(2-hydroxyethyl)histidine; 4) histidine.

402 Osterman-Golkar et a1

C

31

30

29

28

27

26

n - HOEtGua Gua

n ---.-..L.......

170

4 -.... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

.....

........ . . . . . . . . . . . . . . . . . . . . --L ...,

190

33 .........

2

1

rnl M HCl

Fig. 3. Determination of the degree of alkylation of guanine-N-7 in testis DNA. Before counting the N-7-hydroxyethylguanine (HOEtGua) fractions were separated a second time from guanine on the Aminex column. Each fraction was counted for 840 min, subdivided in nine runs. Using the three preceding and the two following fractions as standard with an average of 26.8 cpm, the three HOEtGua fractions were found to contain 9 cpm altogether. The accuracy of this value is estimated from the variance of the standard and of individual samples. From these data the 95 % confidence interval of the excess radioactivity in the HOEtGua fractions is computed to 9.0 rt 1 .1 cpm.

TABLE I. Degree of Alkylation of Hemoglobin (total), Histidine-N7 in Hemoglobin, and Guanine- N-7 in DNA From Livers and Testes Following Treatment of Fischer Rats With Radiolabeled Ethylene Oxide and the Corresponding Doses Calculated According to Equation 1

Analyzed Nucleophilic amount target molecule (mg)

Experiment A 1 Hemoglobin 25

Histidine-N7 25

Guanine-N-7 13

Guanine-N-7 2.9

(total)

in hemoglobin

in liver DNA

in testes DNA

Radioactivity in analyzed

peak (sample) (dim) . .

7,100

83

240

18

Experiment A2 Hemoglobin 19.6 2,400

Guanine-N-7 25 227

Guanine-N-7 15 49

(total)

in liver DNA

in testes DNA

Degree of alkylation

(mol HOEtlg Hb or g DNA, respectively)

120 . 10- '0

1.4 .lo-'"

7.4

2.5 .

25 .lo- l o

1.9 . lo-"

0.67. lo-"

Dose (M h)

5.2 .lo-'

5.2 .

1.4

2.5

1.1 .10-6

1.9 .

0.67. lo-'

Dosimetry of Ethylene Oxide 403

A2 (2.77 * mol kg-I) thus correspond to 14 ppm h and 1.9 ppm h. The degree of alkylation of histidine-Nr in hemoglobin per pprn h of ethylene oxide would be 9.7 * lo-'* mol (g Hb)-' and 15. lopi2 mol (g Hb)-' when estimated from data of experiments A1 and A2, respectively.

Assuming that the clearance of ethylene oxide from tissues follows first-order kinetics, the dose in a tissue may be expressed as the product of the initial concentra- tion of the compound (or the total amount absorbed in the tissue per unit weight), Co, and the mean life, I/X (X = first-order rate constant for the elimination), of its molecules in the tissue, according to

The high value of A, 4.2 h-' when calculated accordingly from experiment A1 using data on the degree of alkylation of hemoglobin and the corresponding rate constant (equation 1); 2.4 h-' when calculated from experiment A2, is similar to the value, 4.6 h-', determined for ethylene oxide in the mouse [Ehrenberg et al, 1974; Oster- man-Golkar et al, 19761 and is also similar to the value estimated for man [Calleman et al, 19781. The corresponding biological half-life of ethylene oxide in the rat, 10 min (exp Al) , 17 min (exp A2) (t% = 0.693/X) is short compared to its half-life in eg water, 76 h at 37°C [cf Osterman-Golkar, 19751, demonstrating an efficient detoxifi- cation of the compound.

The degrees of alky lation of histidine-N. in hemoglobin from chronically ex- posed rats (exp B) are given in Table 11. A good agreement was obtained between determination on the amino acid analyzer and by means of GC-MS. The sensitivity of the former method was insufficient for the determination of the background values (code Nos. 303 and 305). Assuming the degree of alkylation of hemoglobin to increase monotonously with exposure level, known values of exposure levels were assigned to the respective code numbers as given in Table 11. The correctness of this a posteriori assignation was later confirmed by Dr W.M. Snellings (Bushy Run Research Center).

It has been shown experimentally that low degrees of alkylation do not affect the life-span of the erythrocytes and that alkylated (methylated, 2-hydroxyethylated) products of hemoglobin have a high stability in vivo [Osterman-Golkar et al, 1976; Segerback et al, 19781. Therefore, reaction products accumulate in a predictable way (equation 3) during chronic exposure

where a is the increment of the degree of alkylation per unit time and t,, is the life- span of the red cells (60 days in the rat [Berlin, 19641) [Osterman-Golkar et al, 19761.

In the chronic exposures for 6 h per day, 5 days per week, the average daily exposure doses may be computed as (3O.[Et0],,,/7) ppm h where [EtO],,, is the exposure level in ppm. Using equation (3) the average increment of the degree of alkylation per ppm h were calculated at the three exposure levels. The data are presented in Table 111.

Inhalation experiments with mice comprising doses of ethylene oxide from 1.44 ppm h to 41.1 ppm h and experiments where the compound was given by IP injection in amounts from 5.4. mol (kg body weight)-' [Ehrenberg et al, to 840-

404 Osterman-Golkar et al

TABLE 11. Experiment B: N-(2-Hydroxyethyl)Histidine in Hemoglobin Isolated From Rats Exposed to 0, 10,33,100 ppm, 6h/day, 5 daydweek, for 2 Years; 13,380 dpm I4C-Labeled Internal Standard (345 mCi/mmole) Was Added to 0.5 g Hemoglobin Before Hydrolysis

Assigned Radioactivity in nmol HOEtHis exposure

Code nmol HOEtHis in analyzed sample per g of level No." analyzed sample (dpm) hemoglobin (PPW

30 I 1.7 1,302 35 33 302 1.1 349 85 100

0 303 Not detectable -

304 - 0.4 857 - 13 10 0 305 Not detectable -

Analysis on a Biotronic LC 6000 E amino acid analyzer

Analysis by GC-MS 30 1 1.42 1,103 34 33 302 1.17 379 82 100 303 - 0.048 1,019 - 1.3 0 304 0.45 867 14 10 305 0.040 388 2.8 0

"Red cells from three to four animals pooled.

TABLE 111. Estimated Increments of the Degree of Alkylation of Histidine-NT per ppm h of Ethylene Oxide

Ethylene oxide exposure

Experiment A: Experiment B: Degree of alkylation Increment

corresponding to" (30 h/week) to (mol (g Hb)-') (mol (g Hb)- I)

1.9 ppm h 0.29 lo-'' 15 . lo-"

Injected amount Chronic exposure of histidine-N Per PPm h

14 ppm h 1.4 . lo-'' 9.7 . 10-12 10 PPm 14 . lo-' 9.3 . 1 0 - ' 2 b

33 PPm 34 ' 10-9 7.5 ' 10-'2h 100 PPm 82 . lo-' 6.8 .

'For calculation see the text. 'Corrected for background level ( - 2 nmol (g Hb)- ').

1974; Osterman-Golkar et al, 1976; Segerback, 19831 are consistent with a linear relationship between exposure dose and in vivo dose. The data obtained in the present study with the Fischer 344 rat indicate a deviation, although not large, from linearity with a relatively higher rate of detoxification at high levels of ethylene oxide. It has to be pointed out that the estimation of the degree of alkylation per ppm h from experiments A1 and A2 makes use of the minute volume of the rats, a constant with a relatively large uncertainty. There is no obvious explanation to the difference observed between experiments A1 and A2. The deviation from linearity observed in experiment B might be real, reflecting an induction of detoxification enzymes, or a lower rate of uptake at high levels due to tissue damage or anesthesia (exposure levels close to 100 ppm have been found to impair growth) [Hollingsworth et al, 19561.

The application of the obtained data will have to await publication of the biological observations made in the long-term experiment and, if possible, compara- ble data for radiation effects in the Fischer rat.

Dosimetry of Ethylene Oxide 405

ACKNOWLEDGMENTS

Thanks are due Dr. Snellings, Carnegie-Mellon Research Laboratories, Pitts- burg, for providing the hemoglobin samples from chronicaIly exposed rats. The investigation was supported by the Swedish Work Environment Fund and the Swedish Natural Science Research Council. The amino acid analyzer was obtained through a grant from the Knut and Alice Wallenberg Foundation.

REFERENCES

Aaron CS: Molecular dosimetry of chemical mutagens. Selection of appropriate target molecules for

Anson ML, Mirsky ME: Protein coagulation and its reversal. The preparation of insoluble globin,

Berlin NI: Life span of the red cell. In Bishop C, Surgenor DM (eds): “The Red Blood Cell.” New

Brookes P, Lawley PD: The alkylation of guanosine and guanylic acid. J Chem SOC 3923-3928, 1961. Calleman CJ, Wachtmeister CA: Synthesis of N-- and N’-(2-hydroxyethyl)-L-histidines. Acta Chem

Scand B 33:277-280, 1979. Calleman CJ, Ehrenberg L, Jansson B, Osterman-Golkar S, Segerback D, Svensson K, Wachtmeister

CA: Monitoring and risk assessment by means of alkyl groups in hemoglobin in persons occupationally exposed to ethylene oxide. J Environ Pathol Toxicol2:427-442, 1978.

Ehrenberg L: Risk assessment of ethylene oxide and other compounds. In McElheny VK, Abrahamson S (eds): “Banbury Report 1, Assessing Chemical Mutagens: The Risk to Humans.” Cold Spring Harbor, New York: Cold Spring Harbor Laboratory, 1979, pp 157-190.

Ehrenberg L: Methods of comparing risks of radiation and chemicals. The rad equivalence of stochastic effects of chemicals. In “Radiobiological Equivalents of Chemical Pollutants. ” Vienna: IAEA,

Ehrenberg L, Hiesche KD, Osterman-Golkar S, Wennberg I: Evaluation of genetic risks of alkylating agents: Tissue doses in the mouse from air contaminated with ethylene oxide. Mutat Res 24:83- 103, 1974.

Ehrenberg L, Osterman-Golkar S: Alkylation of macromolecules for detecting mutagenic agents. Teratog Carcinog Mutagen 1 : 105-127, 1980.

Hollingsworth RL, Rowe VK, Oyen F, McCollinter DD, Spencer HC: Toxicity of ethylene oxide determined on experimental animals. Arch Ind Health 13:217-227, 1956.

Lawley PD, Thatcher CJ: Methylation of deoxyribonucleic acid in cultured mammalian cells by N- methyl-”nitro-N-nitrosoguanidine. Biochem J 1 I6:693-707, 1970.

Lee WR: Molecular dosimetry of chemical mutagens, determination of molecular dose to the germ line. Mutat Res 38:3 11-3 16, 1976.

Lumb WV: “Small Animal Anesthesia. Philadelphia: Lea and Febiger, 1963. Osterman-Golkar S: Studies on the reaction kinetics of biologically active electrophilic reagents as a

basis for risk estimates. Thesis, Stockholm University, 1975. Osterman-Golkar S, Ehrenberg L, Segerback D, Hallstrom I: Evaluation of genetic risks of alkylating

agents. 11. Haemoglobin as a dose monitor. Mutat Res 34: 1-10, 1976. Segerback D: Alkylation of DNA and hemoglobin in the mouse after exposure to ethene and ethene

oxide. Chem-Biol Interact (in press). Segerback D, Calleman CJ, Ehrenberg L, Lofroth G, Osterman-Golkar S: Evaluation of genetic risks of

alkylating agents. IV. Quantitative determination of alkylated amino acids in haemoglobin as a measure of the dose after treatment of mice with methyl methanesulfonate. Mutat Res 49:71-82, 1978.

determining molecular dose to the germ line. Mutat Res 38:303-310, 1976.

soluble globin and heme. J Gen Physiol 13:469-476, 1930.

York: Academic Press, 1964, pp 423-450.

1980, pp 11-21.