Toxicology, 76 (1992) 49-57 Elsevier Scientific Publishers Ireland Ltd.
The effect of ethylene glycol monomethyl ether and diethylene glycol monomethyl ether on hepatic -r-glutamyl transpeptidase Toshihiro Kawamoto a, Koji Matsuno a, Fujio Kayama a, Keiichi Arashidani b, Masahiro Yoshikawa b and Yasushi Kodama a aDepartment of Environmental Health and bSchool of Nursing and Medical Technology, University of Occupational and Environmental Health, UNIV OEH SCH MED PO ORIO, Yahatanishi ku, Kitakyushu 807 (Japan) (Received April 21st, 1992; accepted July 16th, 1992)
Summary In this paper, we determined whether ethylene glycol monomethyl ether (EGME) and diethylene glycol monomethyl ether (diEGME) induce hepatic 7-glutamyl transpeptidase activity. Male adult Wistar rats weighing 220 g were used as experimental animals. EGME (100, 300 mg/kg per day) and diEGME (500, 1000, 2000 mg/kg per day) were administered by gavage for 1, 2 or 5 days or 4 weeks. In the 4-week study, experimental animals were administered EGME or diEGME once a day orally, 5 days/week. EGME treatment increased the serum 3,-glutamyl transpeptidase (GGT) level significantly, however, diEGME did not. The activities of three other enzymes (SGOT, SGPT and ALP) in serum were not altered by EGME or diEGME treatment and thus there was no biochemical indices of hepatic damage by EGME or diEGME. EGME treatment increased the GGT activities in the liver and lungs. Of the organs examined, the induction of GGT was the greatest in the liver. The inducibility in the liver was 216% for the 5-day treatment and 460% for the 4-week treatment. A dose-dependent increase of hepatic microsomal GGT activity by EGME was observed. On the other hand, renal GGT activities were declined to 72% and 60% of control by the 5-day and 4-week EGME treatments, respectively. DiEGME did not affect the GGT activities in any of the tissues except those of the brain. In the histochemical study, most hepatocytes at the periportal zones were stained with GGT staining after the 4-week treatment. However, the hepatocytes at the central zones were negative.
Key words:7-Glutamyl transpeptidase; Ethylene glycol monomethyl ether; Diethylene glycol monomethyl ether; Histochemistry; Enzyme induction; Glycol ethers
Introduction E t h y l e n e glycol m o n o m e t h y l e t h e r ( E G M E ) is o n e o f a class o f c h e m i c a l s exh i b i t i n g the p o l a r p r o p e r t i e s o f a l c o h o l s a n d t h e n o n - p o l a r p r o p e r t i e s o f ethers.
Correspondence to: Toshihiro Kawamoto, M.D., Ph.D., Department of Environmental Health, University of Occupational and Environmental Health, UNIV OEH SCH MED PO ORIO, Yahata nishi ku, Kitakyushu 807, Japan. 0300-483X/92/$05.00 © 1992 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
50 EGME is used in the photographic, plastic and dyeing industries and as a de-icing additive in military jet fuel . "r-Glutamyl transpeptidase (GGT) is a membranebound enzyme which catalyses the transfer of the ~,-glutamyl moiety of glutathione and other glutamyl peptides to a variety of amino acids . Even though adult rat serum and liver have low levels of GGT activities, it is well known that alcohols and some carcinogenic chemicals induce GGT in liver [3,4]. EGME is metabolized by two major pathways. One involves alcohol and aldehyde dehydrogenase, resulting in the production of methoxyacetic acid. The other competing pathway involves an O-demethylation reaction, producing the formaldehyde and ethylene glycol . Ethylene glycol can be further metabolized to glycolate by alcohol/aldehyde dehydrogenase enzymes . It is suspected that EGME induces hepatic GGT activity because EGME and its metabolite, ethylene glycol, are substrates of alcohol/aldehyde dehydrogenase enzymes. However, there have been no reports regarding the induction of hepatic GGT by glycol ethers. In our experiments we investigated the effect of EGME on hepatic GGT and we also examined the effect of a structural homologue, diethylene glycol monomethyl ether (diEGME) on GGT. Materials and methods Chemicals
Ethylene glycol monomethyl ether (purity: >99%) and diethylene glycol monomethyl ether (purity: > 98%) were purchased from Wako Chemical Co. These chemicals were administered orally to animals after distillation. L-q~-Glutamyl-pnitroanilide was purchased from Sigma Chemical Co., St. Louis, MO. Animal treatment
Male adult Wistar rats (purchased from Kyudo Co., Ltd. Japan) weighing 220 g were used. They were allowed free access to food (CE-2, Clea Japan Inc.) and water and were kept at 24°C on a 24-h light/dark cycle for the duration of the study. EGME (100, 300 mg/kg per day) and diEGME (500, 1000, 2000 mg/kg per day), diluted in distilled water, were administered by gavage for 1, 2 or 5 days or 4 weeks. In the 4-week treatment, the experimental animals were administered EGME or diEGME once a day orally, 5 days/week. Control rats were administered an equivalent volume of distilled water in the same way. Biochemical measurement
The experimental animals were sacrificed 24 h after the final administration. Blood was drawn from the inferior vena cava. Serum alanine aminotransferase (SGOT) and aspartate aminotransferase (SGPT) were determined using TA-L TEST SR-3500, Mizuho Medy, Saga, Japan. Serum alkaline phosphatase (ALP) was assayed using ALP TEST SR-1200, Mizuho Medy, Saga, Japan. The liver, brain, lung, spleen, pancreas and kidney tissues were homogenized at 4°C in a PotterElvehjem homogenizer equipped with a Teflon pestle with 10 volumes of 0.01 M Tris-HCl buffer (pH 8.0) containing 0.15 M NaC1 . Liver microsomes were isolated by the method of van der Hoeven and Coon . GGT enzyme activities of each tissue and blood were assayed using 3,-GTP KIT (SR-1600) supplied by Mizuho Medy, Saga, Japan.
Histochemistry The histochemical study of G G T in the liver was performed using the method of Rutenburg et al. , which utilizes L-3,-glutamyl-p-nitroanilide as a substrate. Statistical analysis Statistical differences between the control group and the treated groups were determined using Tukey's multiple-comparison method . The levels of significant differences were P < 0.01 and P < 0.05. Results
The changes of GGT activities in blood and organs Table I shows serum GGT, SGOT, SGPT and ALP levels in the rats treated with E G M E (300 mg/kg per day) or diEGME (1000 mg/kg per day) for 4 weeks. E G M E treatment increased serum G G T levels significantly, however, diEGME did not. The activities of the other three enzymes (SGOT, SGPT and ALP) in serum were not altered by either E G M E or diEGME treatment. The G G T activities in the various tissues were also examined (Table II). E G M E treatment increased the G G T activities in the liver and lungs. The liver showed the most induction of G G T activity of all the tissues which we examined. The inducibility in the liver was 216% for the 5-day treatment and 460% for the 4-week treatment. The G G T activities in the brain and pancreas were not affected by E G M E treatment. In the kidneys, G G T activities were reduced to 72% and 60% of control by the 5-day and 4-week treatments, respectively. On the other hand, diEGME did not affect the G G T activities in the various tissues, except for a 50% increase in the brain. Effect on the hepatic microsomal GGT activity As it is well known that G G T in liver is mainly located in the microsomes of liver cells , the time course study on hepatic microsomal G G T induction by EGME was performed (Fig. 1). E G M E (300 mg/kg) was administered orally to rats in the same manner for 4 weeks. The significant increase of G G T activity was recognized after the 5-day treatment. After 4 weeks, the G G T activity was 4.5 times
TABLE I S E R U M GGT, SGOT, SGPT A N D A L P LEVELS O F THE RATS T R E A T E D W I T H E G M E (300 mg/kg per day) OR DI E G M E (1000 mg/kg per day) F O R 4 W E E K S (n = no. or animals) Control ( n = 5) G G T (mU/ml) SGOT (IU/I) SGPT(IU/1) A L P (K-A unit)
1.55 81.8 16.6 13.8
Mean and S.D. are represented. *P < 0.05 compared with control.
EGME (n = 5) 0.52 14.6 2.6 2.4
2.43 82.9 21.1 10.6
di E G M E (n = 5) 0.59* 29.1 6.2 3.1
1.99 84.1 15.5 13.6
4- 0.67 4- 25.8 4- 8.8 ± 3.5
52 T A B L E II EFFECTS O F E G M E O R D I E G M E T R E A T M E N T O N T H E G G T ACTIVITIES O F V A R I O U S TISSUES IN R A T S (n = no. of animals) Organs
E G M E (300 mg/kg per day) d i E G M E (1000 mg/kg per day)
5-day treatment Liver Brain Lung Spleen Pancreas Kidney
(n = 6) 0.349 ± 0.912 a0.816 ± 0.617 ± 107.4 ± 598.3 ±
0.026 a 0.101 0.121 0.082 10.5 69.5
(n = 6) 0.757 ± 0.023 (216%¢) ** 0.934 ± 0.071 (102) 1.084 ± 0.052 (133)** 0.853 ± 0.100 (138)** 96.0 ± 18.5 (89) 428.5:1:62.9 (72)**
4-week treatment Liver Brain Lung Spleen Pancreas Kidney
(n = 5) 0.405 ± 1.046 ± 1.058 ± 0.790 ± 113.8 ± 614.9 ±
0.099 0.229 0.153 0.112 15.6 74.4
(n = 5) 1.861 q1.193 ± 1.454 ± 0.839 ± 125.8 q367.2 ±
(n = 5) 0.516 ± 1.645 ± 1.150 ± 0.750 ± 116.0 ± 564.9 ±
0.710 (460)** 0.144 (114) 0.093 (137)** 0.072 (106) 7.8 (111) 74.9 (60)**
0.155 (127) 0.126 (157)** 0.138 (109) 0.137 (95) 13.4 (102) 81.7 (92)
aMean ± S.D. (Unit/g tissue). bnot determined. Cpercentage of control G G T activity. *P < 0.05; **P < 0.01 compared with control.
Fig. 1. Time course of hepatic microsomal G G T activities from rats treated with vehicle (control), E G M E (300 mg/kg) and d i E G M E (2000 mg/kg). Mean and S.D. are represented. *P < 0.05 compared with control. **P < 0.01 compared with control.
53 higher than that of the control. Figure 2 shows the dose-response study on G G T induction. When rats were administered 100 mg/kg and 300 mg/kg E G M E for 4 weeks, the G G T activity was 13.2 q-3.8, 16.7 ± 3.1 mU/mg protein, respectively. On the other hand, diEGME did not cause a significant increase of hepatic microsomal G G T activity, except for the rats which were administered diEGME 2000 mg/kg per day for 4 weeks.
Histochemical study on hepatic GGT induction The distribution of G G T in the liver was investigated using histochemical methods. In the livers of the control rats (Fig. 3a), only the biliary duct epitherium exhibited G G T activity and all the hepatic cells were negative. After the 5-day treatment with 300 mg/kg EGME, G G T activity was detected in some hepatocytes which were adjacent to the portal areas (Fig. 3b). After the 4-week treatment, most hepatocytes at the periportal zones were stained with G G T staining. However, the hepatocytes at the central zones were negative (Fig. 3c). Neither mitotic figures nor disorders in the lobular architecture were observed in the liver of rats treated with E G M E (300 mg/kg per day) for 4 weeks. Discussion In this study, we administered 100 or 300 mg/kg per day of E G M E or 500, I000 or 2000 mg/kg per day of diEGME to rats for 1, 2 or 5 days or 4 weeks. The toxicological effects of these doses were already reported . The atrophies of thymus
GGT activity (rnU/mgprotein) 0 Control
diEGME 500mg/Kg ~
** (5) *
Fig. 2. Dose-responsestudy of hepatic microsomalGGT activitiesfrom rats treated with vehicle(control), EGME and diEGME for 4 weeks. Mean and S.D. are represented. *P < 0.05 compared with control. **P < 0.01 compared with control.
Fig. 3. G G T staining of the liver section from control rat (a), E G M E (300 mg/kg per day, o.p.) treated rat for 5 days (b) or for 4 weeks (c).
55 and testis were remarkable. However, the loss of body weight and liver weight were slightly observed only in the cases of the 300 mg/kg EGME and the 2000 mg/kg diEGME treatments for 20 days. In this study SGOT, SGPT and ALP levels did not indicate any liver damage and H.E. staining of the liver section did not show either an increase in the level of cell proliferation or disorder in the lobular architecture of the liver. We believe that no significant liver damage was brought about by EGME or diEGME treatment in this experiment. It is well known that both serum and hepatic GGT activities increase in patients with chronic alcoholism. Ishii et al.  reported the enhancement of hepatic GGT activity in the liver from chronic alcoholic rats which had been fed a liquid diet containing ethanol for 6 weeks. However, the inducibility of GGT was only twofold. There was no significant enhancement of the enzyme activities of the other organs, except those of the small intestine. They also reported that there was no significant enhancement of GGT activities in the rats treated with ethanol for short periods. We also made the same observations (data not shown). On the other hand, some hepatocarcinogens also induce the hepatic GGT activity. Fiala and Fiala  reported that a single large dose of strong hepato-carcinogens such as dimethylnitrosoamine, 2acetylaminofluorene (2-AAF) or 3'-methyl-4-dimethylaminoazobenzene (3'-MeDAB) led to higher levels of GGT within 24-48 h and that feeding a diet containing carcinogenic 3 '-Me-DAB or 2-AAF increased GGT activity about 10 times higher than that of control rat liver. In our study, we demonstrated an increase of hepatic GGT after EGME treatment. The 5-day administration increased GGT activity 1.9 times and the activity was increased 4.5 times by the 4-week administration. This induction rate is higher than that by ethanol  and lower than by chemical carcinogens . The results of our histochemical study demonstrated an enhancing effect of EGME on GGT activity of rat periportal hepatocytes. The mechanism of induction of GGT and its function in EGME-treated periportal hepatocytes is not clear from our study. Among xenobiotics other than EGME, ethanol , ethynylestradiol , butylated hydroxytoluene [15,16] and phenobarbital  have been shown histochemically to induce GGT activity in rat periportal hepatocytes. Ethanol, butylated hydroxytoluene and phenobarbital are suspected to be cancer promoters. On the other hand, some carcinogens such as N-2-fluorenylacetoamide, cause GGTpositive hepatocellular altered foci in liver . In the liver from rats treated with EGME, there were no such foci. Philologically there are no carcinogenicity data for EGME, and no adequate long-term animal studies have been reported to date. The mutagenicity of EGME has been investigated in a range of in vivo systems using bacteria and mammalian cells. Most studies, except those using very high concentrations of EGME in CHO cells, yielded negative results . The promoting action of EGME is still unknown. It might be important to clarify whether EGME is carcinogenic under an adequate long-term exposure experiment. The GGT activation by diEGME treatment was also examined in our experiment. DiEGME did not induce GGT activity except for the 2000 mg/kg per day diEGME treatment for 4 weeks. EGME and diEGME are structural homologues, however, the induction of hepatic GGT activity was quite different between EGME and diEGME. Kawamoto et al.  reported that EGME treatment increased hepatic alcohol dehydrogenase activity and diEGME induced hepatic cytochrome P-450.
56 T h e r e f o r e , it is s u s p e c t e d t h a t the m e t a b o l i z i n g e n z y m e s i n v o l v e d in the m e t a b o l i s m o f these t w o h o m o l o g u e s m a y be different. F r o m this s t u d y we h a v e c o n c l u d e d t h a t E G M E i n d u c e s h e p a t i c G G T a c t i v i t y dose dependently. On the other hand, diEGME did not induce hepatic GGT activity e x c e p t f o r e x t r e m e l y h i g h doses. I n the h i s t o c h e m i c a l study, E G M E t r e a t m e n t inc r e a s e d G G T a c t i v i t y in t h e p e r i p o r t a l h e p a t o c y t e s w i t h o u t a n y i n c r e a s e in the level o f cell p r o l i f e r a t i o n o r d i s o r d e r in the l o b u l a r a r c h i t e c t u r e o f the liver.
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