Species differences in testicular and hepatic biotransformation of 2-methoxyethanol

Toxicology 96 (1995)2 17-224

ELSEVIER

Species differences in testicular and hepatic biotransformation of 2-methoxyethanol Mary Treinen Moslen* a, Lata Kaphaliaa, Hariharan Balasubramaniana, Yong-Mei Yin”, William W. Aub aDepartment bDepartment of Preventive

of Pathology,

University of Texas Medical Branch, Galveston,

Medicine and Community

TX 77550, USA

Health, University of Texas Medical Branch, Galveston,

TX 77550, USA

Received 15 February 1994; accepted 30 June 1994

Abstract Biotransformation

of 2-methoxyethanol

(2-ME) by alcohol and aldehyde dehydrogenases is an established factor capacity for 2-ME biotransformation by testis or other target tissues. We detected appreciable capacity for 2-ME biotransformation by alcohol dehydrogenase in testes from Sprague-Dawley rats. However, kinetic analysis showed a 6-fold lower affinity for 2-ME by alcohol dehydrogenase of testis compared to liver. 2-ME biotransformation was also detected in testes from Wistar rats and one strain of mice but not in testes from hamsters, guinea pigs, rabbits, dogs, cats or humans. Testes from all these species readily converted the aldehyde metabolite of 2-ME to 2-methoxyacetic acid. Hepatic capacities for 2-ME biotransformation by alcohol dehydrogenase varied from 22 to 2.5 Fmol/mg prot/min with a species rank order of: hamsters > rats = mice > guinea pigs = rabbits. There was no consistent concordance between activities for 2-ME versus ethanol, the prototype substrate for alcohol dehydrogenase, which could reflect substrate preferences of different isozymes. Species differences between rats and hamsters were also found for testicular and hepatic biotransformation of the glycol ethers, 24hoxyethanol and 2-butoxyethanol. Although species differences in capacity for 2-ME biotransformation were found, the observations do not provide an explanation for reported species and strain differences in susceptibility to 2-ME toxicity. in the toxicity of this useful solvent.

Little is known about potential

Keywords: 2-Methoxyethanol; Testicular biotransformation; Ethoxyethanol; 2-Butoxyethanol

Alcohol dehydrogenase;

Aldehyde dehydrogenase;

2-

1. Introduction

2-methoxyethanol; 2-MAld, 2methoxyacetaldehyde; 2-MAA, 2-methoxyacetic acid; 2-EE, Z-ethoxyethanol; 2-BE, 2-butoxyethanol * Corresponding author. Abbreviations:

2-ME,

2-Methoxyethanol(2-ME, omethyl ether) was widely paints, lacquers, epoxyresins coatings. Use of 2-ME as a

0300-483X/95/$09.50 0 1995 Elsevier Science Ireland Ltd. All rights reserved SSDI 0300-483X(94)02921-G

ethylene glycol monused as a solvent in and other protective solvent in household

M. T. Moslen et al. / Toxicology 96 (1995) 217-224

218

2-methoxyethanol

,--&W alcohol dehydrogenaae

I-methoxyacetaldhyde NAD aldehyde dehydrogenaee 2-methoxyacetic

~

J acid

Fig. 1. Proposed scheme for the relationship between the toxicity of 2-methoxyethanol (2-ME) and its sequential biotransformation to 2-methoxyacetaldehyde (2-MAld) and 2-methoxyacetic acid (2-MAA).

and industrial products has declined except for some regions (Lin and Chen, 1993) and specialized purposes such as manufacture of semiconductors (Lamm et al., 1994). Animal studies have demonstrated that 2-ME causes testicular atrophy, bone marrow suppression, teratogenicity, and is a potent immunotoxin (Nagano et al., 1979; Miller et al., 1983; Smialowicz et al., 1991). Shipyard painters exposed to 2-ME and 2-ethoxyethanol(2EE) were found to have lower sperm counts (Welch et al., 1988). As shown in Fig. 1, the toxicity of 2-ME is considered due to its biotransformation to aldehyde (2-MAld) and then acid (2-MAA) metabolites. Specifically, administration of 2-ME concurrently with an inhibitor of alcohol dehydrogenase reduced the testicular atrophy, teratogenicity and immunotoxicity of 2-ME, while administration of 2-MAA caused toxicity to these sites (Moss et al., 1985; Ritter et al., 1985; Smialowicz et al., 1991). MAld was recently found to be immunosuppressive in the rat (Smialowicz et al., 1993) and to cause a dose-dependent mutagenic response in Chinese hamster ovary AS52 cells (Ma et al., 1993). Biotransformation of 2-ME by experimental animals has been studied by many investigators (Moss et al., 1985; Sleet et al., 1986; Medinski et al., 1990; Sabourin et al., 1992; Sumner et al., 1992). However, nearly all have been in vivo studies which characterized the metabolites excreted in urine or the tissue distribution of label derived from radioactive 2-ME. Thus little is known about which tissues are capable of converting 2-ME to its aldehyde and acid metabolites. Of particular in-

terest is the possibility of biotransformation by the testis since this target site of 2-ME is known to have both alcohol and aldehyde dehydrogenases in several species (Messiha, 1981; Rout and Holmes, 1985; Chiao and Van Thiel, 1986; Dafeldecker and Vallee, 1986; Keung, 1988). Our major objective was to investigate the biotransformation of 2-ME by alcohol dehydrogenase and of 2-MAld by aldehyde dehydrogenase in testes from eight species. In addition, hepatic biotransformation capacities were compared in five of the species. 2. Materials and methods 2.1. Sources of testes and livers Cat and dog testes were collected during castration of sexually mature animals (< 6 years old) at a local veterinary hospital. Human testes were obtained at autopsy from non-infectious cases of males less than 45 years old who were autopsied within 12 h of death. Collection of human tissues was done with approval of the University of Texas Medical Branch Institutional Review Board. Testes and livers were obtained from untreated, sexually mature rats, mice, hamsters, guinea pigs and rabbits at the time of sacrifice for other purposes. The only animals ordered for the sake of the experiments reported in this study were Swiss CD1 and C3H strain mice. Sprague-Dawley rats of 250-300 g were from Harlan (Indianapolis, IN). Wistar rats of 230-250 g, Swiss Webster mice of 25-34 g, B6C3-Fl mice of 8-10 weeks, Swiss CD1 (ICR) mice of 6 months, C3H mice of 6 months, and Syrian Golden hamsters of 6-8 weeks were

M. T. Moslen et al. /Toxicology

from Harlan (Houston, TX). Strain 13 Guinea pigs of 350-600 g were from Crest Caviary (Mariposa, CA). New Zealand White rabbits of 2-3 kg were from Myrtles Rabbitry (Thompson, TN). Livers from all species and testes from most species were rapidly frozen on dry ice and stored at -80°C. Testes from dogs and cats were initially frozen at -20°C and then within 1 week were transferred to a -80°C freezer. Preliminary studies indicated that activities of alcohol and/or aldehyde dehydrogenases in testes from rats and humans were remarkably stable even when tissues were kept at room temperature for 12 h before freezing (Y.-M. Yin and M. T. Moslen, unpublished observation). 2.2. Chemicals 2-Methoxyethanol (99% purity), 2-ethoxyethanol (99% purity), 2-butoxyethanol (99% purity), and propionaldehyde (99% purity) were purchased from Aldrich Chemical Company (Milwaukee, WI). 2-Methoxyacetaldehyde was a gift from BASF Corporation (Parsippany, NJ). Ethanol was from Aaper Alcohol and Chemical Company (Shelbyville, KY). All other chemicals were obtained from Sigma Chemical Company (St. Louis, MO). 2.3. Biotransformation assays Testes and livers were homogenized at 4°C in 4 ~01s. of 0.05 M Tris-HCl buffer (pH 8.4) containing 0.1 mM dithiothreitol which other investigators have used to stabilize testicular alcohol dehydrogenase (Yamauchi et al., 1988). The homogenate was centrifuged at 4°C at either 1000 rev./min or 9000 rev./min for 20 min in a Savant High Speed microcentrifuge. The 9000 rev./min supernatant was used for alcohol dehydrogenase assays. The 1000 rev./min supernatant was used for aldehyde dehydrogenase assays since this activity is found in mitochondria as well as microsomes and cytosol (Lindahl and Evces, 1984). Preliminary studies indicated that these were appropriate preparations for analyses of these enzymes in testis. The supernatants were stored at -80°C until used for enzyme assays. Alcohol dehydrogenase activity was assayed kinetically by following the change in NAD ab-

96 (1995) 217-224

219

sorption at 37°C according to the method of Crow et al. (1977). The reaction mixture contained 1.O M Tris-HCl (pH 7.2) (which serves as a buffer and as a trapping agent for aldehydes), 2.8 mM NAD, 18 mM ethanol or 36 mM 2-ME as substrate, and variable amounts of 9000 rev./min supernatant from testes or liver. Aldehyde dehydrogenase activity was assayed kinetically by following the change in NAD absorption at 25°C according to Lindahl and Evces (1984). The reaction mixture contained 20 mM sodium phosphate buffer (pH 8.5) containing 0.33 mM mercaptoethanol, 0.5 mM pyrazole (to inhibit alcohol dehydrogenase), 2 PM rotenone (to inhibit NADH oxidase), 7.5 mM NAD, 6 mM propionaldehyde or 6 mM methoxyacetaldehyde as substrate, and varying amounts of 1000 rev./min supernatants from testes or liver. Assays were done in duplicate or triplicate with appropriate blanks to correct for substrateindependent change in NAD reduction. Linearity of reaction rate with protein concentration was confirmed for the amount of supernatant used for each assay. Protein was measured according to Lowry et al. (1951) using bovine serum albumin as the standard. Activities are expressed as the rate of NADH formation per min per mg protein. 2.4 Statistical analysis Values are expressed as mean f S.D.except for a few instances where analyses were done on a pool of tissues from multiple animals. Activities towards glycol ether substrates were evaluated by analysis of variance (ANOVA) followed by post hoc comparison of group means according to the Student-Newman-Keuls method. Regression analysis of double reciprocal plots was used to determine the Kmapi,. 3. Results and discussion 3.1. Testicular biotransformation of 2-ME and 2h4Ald

Table 1 compares the testicular biotransformation of 2-ME and 2-MAld by alcohol and aldehyde dehydrogenases of two strains of rats and mice and six other species. Also shown are the activities for ethanol and propionaldehyde which are conven-

M. T. Moslen et al. /Toxicology 96 (1995) 217-224

220

Table 1 Species differences in testicular alcohol and aldehyde dehydrogenase Species

Sprague-Dawley rat Wistar rat Swiss Webster mouse B6C3-FI mouse Syrian Golden hamster Strain I3 guinea pig New Zealand White rabbit Dog Cat Human

N

4 P, P 6 4 3 4 IO IO IO

Alcohol dehydrogenase (pmollmg prot/min)

Aldehyde dehydrogenase (pmollmg prot/min)

Ethanol

2-ME

Propionaldehyde

2-MAld

1.4 f 0.2 I.1 2.3

0.52 f 0.01 0.31 NDb

0.5 Et 0.05

0.13

4.9 f 0.2 ND ND ND ND ND

ND ND ND ND ND ND

4.2 f 0.3 1.8 8.2 7.9 f 1.1 4.6 f 0.2 4.4 f 0.5 8.8 f 0.5 3.1 f 0.6 6.7 f 0.4 1.3 f 0.4

1.6 l 4.0 9.6 1.3 f 7.4 f 8.1 f 9.6 zt 4.0 f 8.9 f 2.0 f

l

0.02

0.2

0.7 0.3 0.1 0.4 I.6 0.5 0.5

Units are means f SD. from a group of the indicated N. ‘Pool of testes from >6 animals. bNot detectable.

tionally used as prototype substrates for these two enzymes. Testes from both Sprague-Dawley and Wistar strains of rats showed appreciable capacity to convert 2-ME to 2-MAld, one mouse strain, the B6C3-Fl, had low activities, while no activity could be detected in any of the other six species. There was no consistent concordance between the testicular activities found for 2-ME compared to ethanol among the species. For example, in Sprague-Dawley rats the activity for 2-ME was

one-third that for ethanol, while Swiss Webster mice and Syrian Golden hamsters had no detectable activities for 2-ME but the highest activities for ethanol. Of particular interest was the lack of detectable activity for 2-ME in hamster testes since the testis of this species has been reported to have an organ specific alcohol dehydrogenase that oxidizes the endogenous alcohol, retinol, very eficiently (Keung, 1988). Thus our results document that the testis of some species has a limited capa-

Table 2 Species differences in hepatic alcohol and aldehyde dehydrogenase Species

Sprague-Dawley rat Swiss CD-I mouse C3H mouse B6C3-Fl mouse Syrian Golden hamster Strain I3 guinea pig New Zealand White rabbit

N

Alcohol dehydrogenase (pmollmg prot/min)

Aldehyde dehydrogenase (pmollmg prot/min)

Ethanol

Propionaldehyde

2-MAld

12.6 f 19.4 f 18.4 f 15.3 f 22.1 f 22.0 f 20.9 f

21.6 f 18.0 f 18.4 f 16.9 f 46.7 f 23.6 f 21.3 f

8.7 f 13.1 f 10.3 f 27.9 f 44.7 f 4.2 f 25.4 f

2-ME 0.9 0.6 0.5 I.4 2.5 I.0 I.0

Units are means f SD from a group of the indicated N.

4.8 5.4 4.1 5.9 22.4 2.9 2.5

f f f f f zt f

0.4 0.6 0.9 0.7 1.5 0.9 0.2

1.2 2.6 1.2 0.8 I.0 2.1 0.4

1.2 3.3 I.1 0.9 1.9 0.7 0.7

221

M. T. Moslen et al. / Toxicology96 (1995) 217-224

city for biotransformation of 2-ME and that the level of this activity is not concordant with that for the prototype substrate ethanol. In contrast to a detectable capacity of testes from only two of the eight species to metabolize 2ME, testes from all species showed readily detectable capacity to biotransform 2-MAld to 2-MAA. There was little relationship among the species between the level of aldehyde dehydrogenase activity for 2-MAld compared to that for the prototype aldehyde substrate, propionaldehyde. Activities for 2-MAld were about twice as high in testes from Sprague-Dawley and Wistar rats, Syrian Golden hamsters and guinea pigs; close to the same for

ALCOHOL _.e .= <

DEHYDROGENASE

1

1.6..

.g ii

1.2..

{

0.8..

3.2. Hepatic

of

2-ME

and

Table 2 compares the hepatic biotransformation of 2-ME and 2-MAld by Sprague-Dawley rats to that in livers from three strains of mice and three other species. Also shown are the hepatic activities for ethanol and propionaldehyde, the prototype substrates for alcohol and aldehyde dehydrogenases. Livers from all species showed substantial levels of activity for 2-ME that ranged

ALCOHOL DEHYDROGENASE _.1.6

2-n

biotrunsformation

2-MAld

ACTIVIM

RAT TESTES * p(D.OSm

Swiss Webster mice, rabbits, dogs, cats and humans; but 4-fold lower for the B6C3-Fl mouse strain.

ACTIVITY

HAMSTER TESTES * p (0.01 n 2-EE

.*

1.2

B

0.0 *

i

+

E

0.4

Oe4:* o.oa

2-m

2-w

2-n

0.0

2-w

substrate (36 mM)

substrate

so-

._

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I

0

6

.s 3

k

z

I

*p
*p(O.O5vs2-EE

30

20

4

s t 2

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(36 mM)

HAMSTER LIVER

RAT LIVER 40..

2-m

2-EE

10 01

0

Z-ME

2-BE

2-n

substrata

(36

mM)

substrata

(36 mM)

Fig. 2. Comparison of the biotransformation of 2-ME, 2-EE and 2-BE by alcohol dehydrogenase in testes from Sprague-Dawley rats and Syrian Golden hamsters (top panels) and in livers from Sprague-Dawley rats and Syrian Golden hamsters (lower panels). Values are means + SD. for four animals per group. Note the higher scale for the hamster liver compared to the rat liver.

222

M. T. Moslen et al. / Toxicology 96 (1995) 217-224

over a factor of 9. Lowest activities were in livers from rabbits and guinea pigs while the highest level was in livers from hamsters. Thus species show substantial differences in hepatic capacity to biotransform 2-ME. As observed with the testis, there was no consistent concordance between the hepatic activities for 2-ME compared to ethanol among the species. Liver from one species, the guinea pig, showed similar activities for 2-ME vs. ethanol, while for all other species the activities for 2-ME were approximately one-half to one-tenth lower. Note the lofold range in the level of hepatic activities for ethanol with the lowest activities in the guinea pig and the highest in the hamster. Further studies are needed to determine if the observed lack of concordance for biotransformation of ethanol versus 2-ME is associated with substrate preferences of the alcohol dehydrogenase isozymes present in testis and liver. Unlike the substantial species differences in hepatic capacity to metabolize 2-ME by alcohol dehydrogenase, hepatic aldehyde dehydrogenase activity for 2-MAld showed modest variation with all values clustered except for the high activity in hamsters. Note that hepatic activities for 2-MAld were appreciably higher than activities for propionaldehyde only in rat and hamster. 3.3. Biotransformation of 2-EE and 2-BE’ Some other glycol ethers also cause testicular atrophy and/or other toxic effects as a consequence of their biotransformation. Administration of 2-EE leads to detrimental effects at the same target sites as 2-ME while 2-butoxyethanol (2-BE) has a more selective effect as a hemolytic agent (Foster et al., 1983; Hardin et al., 1984; Smialowicz et al., 1992b; Tyler, 1984). Therefore, we assessed testicular and hepatic capacities to biotransform 2-EE and 2-BE by alcohol dehydrogenase. These comparisons were done with Sprague-Dawley rats which had the highest observed testicular capacity to biotransform 2-ME and with Syrian Golden hamsters which had no detectable testicular capacity to biotransform 2ME despite high activities for ethanol (Table 1). As shown in the top panels of Fig. 2, testes from rats and hamsters had similar levels of alcohol

dehydrogenase activity for 2-EE, but markedly different activities for 2-ME and 2-BE. Livers from both rats and hamster exhibited highest activities for 2-EE compared to the other two glycol ethers (bottom panels of Fig. 2). However, the rat liver activities towards each substrate were approximately one-fifth that of hamster liver. Only hamster testes showed activity for 2-BE that was higher than that for the other two glycol ethers. Thus, species differences exist in the capacities for testicular and hepatic biotransformation of 2-EE and 2-BE as well as 2-ME. 3.4. Kinetic analysis A particular concern regarding exposures to 2ME is the response to low environmental level exposures by target tissues. In order to determine the

m

.z

12..

3 9,

9..

E $

13-*

:E

3..

Oi -1

0

1 l/

2

3

[ P-mdhoxyethand

-1 /

4

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] mY

.E

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TEslls KmaPP - 0.39

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.

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Fig. 3. Comparisons of the double reciprocal plots for 2-ME (top panel) and ethanol (bottom panel) biotransformation by testis and liver from Sprague-Dawley rats. Each point is the mean of triplicate determinations in a poolof tissue from 5 rats.

MT.

Moslen et al. / Toxicology 96 (1995) 217-224

potential for primary testicular biotransformation of low levels of 2-ME, we compared the substrate affinity of the Sprague-Dawley rat testis versus liver by standard kinetics analysis. As shown in the upper panel of Fig. 3, the Km,, for 2-ME of the testis was 6-fold higher than that of the liver. Testes, however, had a 3-fold lower Km,, for ethanol than liver (lower panel of Fig. 3). Consequently at lower levels of exposure, the capacity of the rat testis for biotransformation of 2-ME would be considerably less efficient than the liver of this species. 3.5 Potential relevance to known differences in 2ME toxicity Our assessment of species and strain differences in hepatic 2-ME biotransformation was largely limited to tissues collected from animals sacrificed for other purposes. Some species and strain differences in susceptibility to 2-ME toxicity have been described, but we are unaware of toxicity studies in Syrian Golden hamsters which we found to have the greatest hepatic capacity for 2-ME biotransformation by alcohol dehydrogenase (Table 2). Smialowicz et al. (1992) reported a marked species variance for young adult female rodents; C57BW6J mice were insensitive to the immunosuppressive effects of both 2-ME and 2-MAA at dose levels which had profound effects on thymus weights and lympho- proliferative functions of Fisher 344 rats. Our data does not provide any insight into this species difference since metabolic capacities were studied only in tissues from male animals and no studies were done on tissues from Fisher 344 rats or C57BW6J mice. We did, however, assess hepatic biotranformation in tissues from male animals reported in two studies to exhibit species or strains differences in response to 2-ME. Miller et al. (1983) observed lymphoid tissue atrophy and testicular degenerative changes after inhalation of a lower dose of 2ME in male New Zealand White rabbits than male Sprague-Dawley rats. Our data does not provide an explanation for this species difference since hepatic alcohol dehydrogenase activity for 2-ME by the more vulnerable rabbit species was about half that for the Sprague-Dawley rat (Table 2). Reproductive studies by Chapin et al. (1993) in-

223

dicated that male C3H mice were more sensitive to 2-ME exposure than male Swiss CD-1 mice in regard to fertility and sperm motility. We found that these mouse strains had similar capacities for hepatic biotransformation of 2-ME and 2-MAld (Table 2). Further studies of alternative metabolic pathways such as dealkylase (Medinsky et al., 1990; Sabourin et al., 1992) and of in vivo pharmacokinetics are needed before metabolic differences can be ruled out as a basis for the reported strain and species differences in 2-ME toxicity. Acknowledgements This project was supported in part by NIEHS grant ES04926 and the John Sealy Foundation. The 2-methoxyacetaldehyde was a gift from the BASF corporation. We thank Dr. Bruce Austin and his staff in the Austin Veterinary Hospital for the cat and dog testes, Dr. Hari Bhat for the hamster testes, Dr.’ Judy Aronson for the guinea pig testes, Dr. Johnny Petersen for the rabbit testes, Dr. Edward Postlethwait for the Swiss Webster mice testes, and Dr. Stephen Wilson for the Wistar rat testes. We thank Dr. Abida Haque and her staff in the UTMB autopsy service for collecting the human testes. References Chapin, R.E., Morrissey, R.E., Gulati, D.K., Hope, E., Barnes, L.H., Russell, S.A. and Kennedy, S.R. (1993) Are mouse strains differentially susceptible to the reproductive toxicity of ethylene glycol monomethyl ether? A study of three strains. Fundam. Appl. Toxicol. 21, 8- 14. Chiao, Y.-B. and Van Thiel, D.H. (1986) Characterization of rat testicular alcohol dehydrogenase. Alcohol Alcoholism 21, 9-15. Crow, K.E., Cornell, N.W. and Veech, R.L. (1977) The rate of ethanol metabolism in isolated rat hepatocytes. Alcoholism: Clin. Exp. Res. I, 43-47. Dafeldecker, W.P. and Vallee, B.L. (1986) Organ-specific human alcohol dehydrogenase: Isolation and characterization of isozymes from testis. B&hem. Biophys. Res. Commutt. 134, 1056-1063. Foster, P.M.D., Creasy, D.M., Foster, J.R., Thomas, L.V., Cook, M. W. and Gangolli, SD. (1983) Testicular toxicity of ethylene glycol monomethyl and monoethyl ethers in the rat. Toxicol. Appl. Pharmacol. 69, 385-399. Hardin, B.D., Goad, P.T. and Burg, J.R. (1984) Developmental

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toxicity of four glycol ethers applied cutaneously to rat. Environ. Health Perspect. 57, 69-74. Keung, W.-M. (1988) A genuine organ specific alcohol dehydrogenase from hamster testes: Isolation, characterixation and developmental changes. B&hem. Biophys. Res. Commun. 156, 38-45. Lamm, S.H., Kutcher, J.S. and Greenblatt, J. (1994) Spontaneous abortions and glycol ethers in the semiconductor industry. A review of two large epidemiological studies. Proceedings of International Symposium on Health Hazards of Glycol Ethers, p. VII 14 Lin, C.-K. and Chen, R.-Y. (1993) Survey of glycol ether use in Taiwan, 1991. Am. J. Ind. Med. 24, 101-108. Lindahl, R. and Evces, S. (1984) Comparative subcellular distribution of aldehyde dehydrogenase in rat, mouse and rabbit liver. B&hem. Pharmacol. 33, 3383-3389. Lowry, O.H., Rosenbrough, N.J., Farr, A.L. and Randall, R.J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-275. Ma, H., An, J., Hsie. A.W. and Au, W.W. (1993) Mutagenicity and cytotoxicity of 2-methoxyethanol and its metabolites in Chinese hamster cells (the CHO/HPRT and AS52/GPT assays). Mutat. Res. 298, 219-225. Medinsky, M.A., Singh, G., Bechtold, W.E., Bond, J.A., Sabourin, P.J., Bimbaum, L.S. and Henderson, R.F. (1990) Disposition of three glycol ethers administered in drinking water to male F344M rats. Toxicol. Appl. Pharmacol. 102, 443-455. Messiha, F.S. (1981) Subcellular fractionation of alcohol and aldehyde dehydrogenasc activity in rat testicles. Prog. Biochem. Pharmacol. 18, 155-166. Miller, R.R., Ayres, J.A., Young, J.T. and McKenna, M.J. (1983) Ethylene glycol monomethyl ether. I. Subchronic vapor inhalation study with rats and rabbits. Fundam. Appl. Toxicol. 3, 49-54. Moss, E.J., Thomas, L.V., Cook, M.W., Walters, D.G., Foster, P.M.D., Creasy, D.M. and Gray, T.J.B. (1985) The role of metabolism in 2-methoxyethanol-induced testicular toxicity. Toxicol. Appl. Pharmacol. 79, 480-489. Nagano, K.. Nakayama, E., Koyano, M., Oobayashi, H., Adachi, H., and Yamada, T. (1979) Testicular atrophy of mice induced by ethylene glycol mono alkyl ethers. Jpn. J. Ind. Health. 21, 29-35. Ritter, E.J., Scott, W.J., Randall, J.L. and Ritter, J.M. (1985) Teratogenicity of dimethoxyethyl phthalate and its metabolites methoxyethanol and methoxyacetic acid in the rat. Teratology 32, 25-31. Rout, U.K. and Holmes, R.S. (1985) Isoelectric focusing stud-

ies of aldehyde dehydrogenases from mouse tissues: Variant phenotypes of liver, stomach and testis isozymes. Comp. B&hem. Physiol. 8 I B, 647-651. Sabourin, P.J., Medinsky, M.A., Thurmond, F., Bimbaum, L.S. and Henderson, R.F. (1992) Effect of dose on the disposition of methoxyethanol, ethoxyethanol, and butoxyethanol administered dermally to male F344/n rats. Fundam. Appl. Toxicol. 19, 124-132. Sleet, R.B., John-Greene, J.A. and Welsch, F. (1986) Localization of radioactivity from f-methoxy[ 1,2- t4C]ethanol in maternal and conceptus compartments of CD-l mice. Toxicol. Appl. Pharmacol. 84, 25-35. Smialowicx, R.J., Riddle, M.M., Luebke, R.W., Copeland, C.B., Andrews, D., Rogers, R.R., Gray L.E. and Laskey, J.W. (1991) Immunotoxicity of 2-methoxyethanol following oral administration in Fischer 344 rats. Toxicol. Appl. Pharmacol. 109,494-506. Smialowicz, R.J., Riddle, M.M., Williams, W.C., Copeland, C.B., Luebke, R.W. and Andrews, D.L. (1992a) Differences between rats and mice in the immunosuppressive activity of 2-methoxyethanol and 2-methoxyacetic acid. Toxicology 74, 57-67. Smialowicz, R.J., Williams, W.C., Riddle, M.M., Andrews, D.L., Luebke, R.W. and Copeland, C.B. (1992b) Comparative immunosuppression of various glycol ethers orally administered to Fischer 344 rats. Fundam. Appl. Toxicol. 18, 621-627. Smialowicz, R.J., Riddle, M.M. and Williams, WC. (1993) Methoxyacetaldehyde, an intermediate metabolite of 2methoxyethanol, is immunosuppressive in the rat. Fundam. Appl. Toxicol. 21, l-7. Sumner, S.C.J., Stedman, D.B., Clarke, D.O., Welsch, F. and Fennell, T.R. (I 992) Characterization of urinary metabolites from [1,2,methoxy-13C]-2-methoxyethanol in mice using “C nuclear magnetic resonance spectroscopy. Chem. Res. Toxicol. 5, 553-560. Tyler, T.R. (1984) Acute and subchronic toxicity of ethylene glycol monobutyl ether. Environ. Health Perspect. 57, 185-191. Welch, L.S., Schrader, S.M., Turner T.W. and Cullen, M.R. (1988) Effects of exposure to ethylene glycol ethers on shipyard painters: II. Male reproduction. Am. J. Ind. Med. 14, 509-526. Yamauchi, M., Potter J.J. and Mezey, E. (1988) Detection and localization of immunoreactive alcohol dehydrogenase protein in the rat testis. Alcoholism: Clin. Exp. Res. 12, 143-146.