Time course of dolichol and dolichyl phosphate during chemical carcinogenesis in rat liver

Biochimica et Biophysics Elsevier

567

Acta 921 (1987) 567-574

BBA 52659

Time course of dolichol and dolichyl phosphate during chemical carcinogenesis in rat liver Kouji Yamada,

Kazuo Tsukidate, Toyohiko Taki, Kouichi and Tadashi Sato Tsukuba Research Laboratories, (Received

Key words:

Dolichol;

Dolichyl

phosphate;

Katayama

Eisai Co., Ltd., Ibaragi (Japan) 7 May 1987)

Hydroxymethylglutaryl-CoA; (Rat liver)

Mannosyl

transferase;

HPLC;

Hyperplastic liver nodules were induced in rats by administration of an initiator (diethylnitrosamine or 3’-methyl-4-dimethylaminoazobenzene) and/or a promoter (phenobarbital) by the method reported by Tatematsu et al. (1983, Carcinogenesis 4, 381-386). The dolichol content in the liver and liver microsomes of the rats treated with the initiator were approx. 15times higher than that of the control and rats treated with only the promoter. However, the composition of dolichols was not changed. The time course of the dolichyl phosphate concentration in the rat liver treated with both initiator and promoter showed a pattern different from that in the control liver, the initiator-treated liver or the promoter-treated liver. The main component of dolichyl phosphate in liver treated with both the initiator and promoter changed from that with 18 isoprene units to that with 19. It is suggested that the changes in liver dolichols and dolichyl phosphates may be related to the formation of hyperplastic liver nodules.

Introduction Dolichols are a group of polyprenols with 16-22 isoprene units which exist ubiquitously in tissues and biological fluids as free alcohols, phosphate esters and esters of fatty acids [l]. In many mammals and yeasts, dolichyl phosphates serve as carriers of saccharide moieties in the transfer of sugars to asparagine residues of polypeptides [2-61. The functions of free dolichols and dolichyl esters, however, have not been established. Since dolichols, cholesterol and ubiquinone are synthesized by a branched pathway from mevalonate

Abbreviation:

HMG-CoA,

3-hydroxy-3-methylglutaryl-CoA.

Correspondence: K. Yamada, Tsukuba Research Laboratories, Eisai Co., Ltd., 1-3, Tokodai-5-chome, Toyosato-machi, Tsukuba-gun, Ibaragi 300-26, Japan.

0005-2760/87/$03.50

0 1987 Elsevier Science Publishers

metabolism or through the major branch-point of farnesyl pyrophosphate, many investigators have been interested in the regulation of dolichol biosynthesis. Dolichol synthesis has been studied in the developing sea urchin embryo [7,8], erythropoietic spleen [9], developing mouse brain [lo] and regenerating rat liver [ll-131. Since dolichyl phosphate is involved as an obligatory lipid intermediate in several steps of glycoprotein synthesis and dolichyl phosphate levels regulate the synthesis of N-linked glycoprotein [14-161, several studies concerning the amount, composition and distribution of dolichols and dolichyl phosphate during chemical carcinogenesis [17] and in tumor cells [18-201 have been reported. Recently, Ito and co-workers have been established a short term in vivo system for the generation of hyperplastic liver nodules by using partially hepatectomized rats [21,22].

B.V. (Biomedical

Division)

568

The time courses of dolichols and dolichyl phosphate in regenerating rat liver have been measured by our highly sensitive HPLC methods [23,24] and have been reported recently [25]. In the present study, we have analyzed the amount and composition of dolichols and dolichyl phosphate during chemical carcinogenesis in the rat short-term in vivo assay system. This paper presents the results, as well as the time courses of HMG-CoA reductase activity and mannosyl transferase activity in the rat short-term in vivo assay system. Materials and Methods Chemicals and reagents. Diethylnitrosamine, 3’-methyl-4-dimethylaminoazobenzene and phenobarbital were purchased from Nakarai Chemical Co. (Kyoto, Japan). GDP[‘4C]mannose and DL-[3-i4C]HMG-CoA were purchased from Amersham Int. (U.K.). Dolichols and dolichyl phosphate homologs used were the preparations described previously [23,24]. Didecaprenylethanol, as an internal standard for dolichols, and 2,2-didecaprenylethyl phosphate, as an internal standard for dolichyl phosphate, were synthesized in our laboratories [23,24]. Anthracene-9-carboxylic acid was purchased from Aldrich Chemical Company. 3-(9-Anthryl)-diazo-2-propene was prepared from 3-(9-anthryl)-propenal by the method of Nakaya et al. [26]. The standard dolichols, dolichyl phosphate and internal standards were dissolved in ethyl ether and stored at - 20 o C. Animals. Male Fisher 344 rats weighing about 150 g were used. Partial hepatectomy of about 70% of the liver was performed under diethyl ether anesthesia by the method of Higgins and Anderson [27]. The animals were killed at the appropriate time after the operation, and the regenerating livers were separated from the non-regenerated portion and were stored frozen at - 80 o C until analysis. Rat short-term assay system. The experiments using the short-term assay system were performed by the method of Ito and co-workers [21,22]. In brief, male Fisher 344 rats were pretreated with a single intraperitoneal injection of diethylnitrosamine (200 mg/kg body weight) or 3’-methyl-4-dimethylaminoazobenzene (600 mg/kg body weight)

as an initiator, and 2 weeks later the rats were given 0.05% phenobarbital in the diet for 6 weeks as a promoter. All animals were subjected to partial hepatectomy 3 weeks after the diethylnitrosamine treatment. Rats of the control group received partial hepatectomy only. Other groups of rats were treated with the initiator only or the promoter only. Preparation of rat liver microsomes. The liver was homogenized in 0.25 M sucrose. The homogenates were centrifuged at 10000 x g for 20 min, and the supernatants were centrifuged at 100000 X g for 60 min. The microsomes thus pelleted were washed by suspending them in 0.15 M TrisHCl buffer (pH 7.9) and centrifuged at 100 000 X g for 60 min. The washed microsomes were resuspended in 0.25 M sucrose. Determination

of dolichols

and

dolichyl phos-

phate.

Dolichols in the liver were subjected to extracted with n-hexane, alkaline hydrolysis, evaporated to dryness and analyzed by HPLC using ultraviolet detection and using of 2,2-didecaprenylethanol as an internal standard. Dolichols in the rat liver microsomes were subjected to alkaline hydrolysis, extracted with n-hexane, derivatized with anthracene-9-carboxylic acid and analyzed by HPLC with fluorescence detection. Dolichyl phosphate homologs in the liver were subjected to mild acid hydrolysis, extracted with diethyl ether, derivatized with 3-(9-anthryl)-diazo2-propene and then determined by HPLC with fluorescence detection, using 2,2_didecaprenylethyl phosphate as an internal standard. The HPLC methods have been reported in detail elsewhere [23,24]. Determination of cholesterol in rat liver. Approx. 100 mg of rat liver was homogenized in 1 ml of distilled water. The homogenate was extracted twice with 4 vol. of chloroform/methanol (2 : 1). The organic phase was evaporated to dryness under N, gas and the residue was dissolved in 1 ml of chloroform/methanol (2 : 1). A 100 pl aliquot of the solution was evaporated to dryness and the residue was dissolved in 100 ~1 of isopropyl alcohol. Cholesterol in the isopropyl alcohol solution was measured by using Iatro Lipo TC (Iatro Inc., Tokyo, Japan), a reagent kit based on the cholesterol oxidase method. Assay

for

transfer

of

[‘4C]mannose

from

569

GDP[‘4C]mannose to lipid intermediates. The reaction mixture in 1 ml consisted of 30 mM mercaptoethanol, 2 mM AMP, 0.5 PCi of GDP[r4C]mannose, and microsomes (1.2 mg protein). The reaction was started by the addition of the radioactive substrate and incubation was performed at 30’ C for 15 min. After the incubation, dolichyl mannosyl phosphate was extracted with chloroform/methanol (2 : and

DL-[3-

r4C]HMG-CoA, microsomes (0.12 protein). reaction was by the of microsomes, continued at for 20 The reaction terminated by addition of ~1 of M HCi, the resulting was

incubated at C for min. Then, mixture was by a chromatographic [28]. Dolichol activity. For ments of monophosphatase activity, pg of dolichyl phosphate diethyl ether 50 ~1 0.1% Triton were mixed dried. The was taken in 65 TrisHCl 6.5) 1 EDTA and mM dithioto give final vol. 50 ~1. mixture was at 37” for 15 after the dition of ~1 microsomes mg protein). reaction was by adding ml of pyrogall01 methanol, 2 of H,O 0.25 ml a 50% aqueous solution. mixture was to alkaline and dolichol tent in was measured the HPLC

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Days after hepatectomy Fig. 1. Time courses of dolichol concentration in the regenerating liver. (A) One (control) group of rats was not treated with chemicals, (0). Other rats were treated with diethylnitrosamine (A). with phenobarbital (A) and with diethylnitrosamine plus phenobarbital (0). Vertical bars, are S.E. of the mean of three separate experiments (B) The rats were treated with 3’-methyl-4-dimethylaminoazobenzene (A), with phenobarbital (A) and with 3’-methyl-4-dimethylaminoazobenzene plus phenobarbital (0). The control rats were not treated (0). Vertical bars, S.E. of the means of three separate experiments.

570

Results Generation of hyperplastic liver nodules On day 35 after partial hepatectomy, rats treated with the initiator and promoter showed a high incidence of hyperplastic liver nodules (numbers of hyperplastic nodules; 28.64 f 4.02/cm2, mean and S.E. of six experiments), which were confirmed by microscopic analysis. When rats were treated with the initiator only, the incidence of the hyperplastic nodules was less than 20% (numbers of hyperplastic nodules; 4.28 + 1.87/cm2, mean and S.E. of six experiments) of that in the group treated with both the initiator and promoter. The control rats and the rats treated with the promoter only developed no hyperplastic liver nodules. These results were in good agreement with the reports of Ito and co-workers [21,22]. Distribution of dolichols in liver and liver microsomes The concentration and composition of dolichols after partial hepatectomy were analyzed by the HPLC method, and the results are shown in Fig. 1. The time course of the dolichol contents in the liver of all groups was in good agreement with our previous results in regenerating rat liver [25]. In all groups, the concentration of liver dolichols decreased gradually during the first 3-5 days after partial hepatectomy, and then from day 5 on increased gradually. However, the dolichol contents in liver treated with the initiator or both the initiator and promoter were 1.5-times higher than those in the control liver and the promoter-treated liver during the experimental period. The composition of dolichols in liver showed no significant difference among the groups during the experimental period. Fig. 2 shows the time course of the dolichol content in the liver microsomes. The time courses were similar to those in the liver and there was no change in the composition of dolichols in the liver microsomes. Distribution of dolichyl phosphate in liver The level of dolichyl phosphate in the liver was analyzed by the HPLC method and the results are shown in Fig. 3. The time course of dolichyl phosphate content in the control liver was in good

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Days after hepatectomy

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Fig. 2. Time courses of dolichol concentration in the regenerating liver microsomes. Dolichol concentration was measured by HPLC as described in Materials and Methods. Conditions (and symbols) as in Fig. 1A.

agreement with that in the regenerating rat liver described previously [25]. The concentration of liver dolichyl phosphate in the control, the initiator-treated or the promoter-treated group increased gradually during the first 5 days after partial hepatectomy, and then decreased gradually. When the rats were treated with both initiator and promoter, the dolichyl phosphate content reached a peak on day 3 and the content on days 3, 14 and 35 were significantly higher than those of the other groups. Furthermore, from days 7-35, the composition of dolichyl phosphate in the initiator plus promoter group showed a change in the relative amount of dolichyl phosphate with 18 and 19 isoprene units, as shown in Table I. Fig. 4 shows mannosyltransferase activity in the liver microsomes. The time course of mannosyltransferase activity was similar to that of the dolichyl phosphate content in the liver.

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Days after hepatectomy Fig. 3. Time courses

of dolichyl

phosphate

concentration Conditions

in the regenerating liver. (A) Conditions (and symbols) as in Fig. 1B.

HMG-CoA reductase activity and dolichol monophosphatase activity Fig. 5 shows the time courses of total cholesterol

TABLE

Control Initiator

as in Fig. IA. (B)

T

I

COMPOSITION OF DOLICHYL PHOSPHATE LIVER ON DAY 35 AFTER HEPATECTOMY Treatment

T

(and symbols)

group

Composition

IN

THE

(Sg)

n=17

18

19

20

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11.8

14.7 14.5

42.1 42.1

31.4 31.7

11.8 11.7

14.2

43.0

30.9

11.9

only

diethylnitrosamine 3’-methyl-4-dimethylaminoazobenzene Promoter only phenob~bital Initiator + promoter diethylnitrosamine + phenobarbital 3’-methyl-4-dimethylaminoazobenzene+phenobarbital

12.6 13.1

34.6 36.8

41.9 39.3

10.9 10.8

Oays after hepatectomy

35 (days)

Fig. 4. Time courses of mannosyltransferase activity in the regenerating liver microsomes. Conditions (and symbols) as in Fig. 1A.

(A)

Fig.

(B)

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14

5. Time courses concentration

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% 0 (days) Days after hepatectomy

of total cholesterol concentration of total cholesterol. (B) The activity

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and HMG-CoA reductase activity in the regenerating liver. (A) The of HMG-CoA reductase. Conditions (and symbols) as in Fig. 1A.

in liver and those of HMG-CoA reductase activity in the liver microsomes. The total cholesterol level in the liver increased during the first 3 days and then rapidly decreased to the normal level. The time course of te HMG-CoA reductase activity in the liver microsomes was similar to that of total cholesterol. Among the groups, no significant difference in the cholesterol level or HMG-CoA reductase activity was found. Fig. 6 shows the dolichol monophosphatase activity in the liver microsomes. No change and no difference among the four groups could be detected during the experimental period. Discussion

Recently, Eggens et al. [17] studied the changes in the amount, composition and distribution of dolichols and dolichyl phosphate during 2acetylaminofluorene-induced hepatocarcinogenesis in rats fed on a diet containing 0.05% 2acetylaminofluorene for 20 weeks. They showed that the dolichol content was increased and the composition was changed in hyperplastic nodules.

In this study, we have studied the changes in the amount and composition of dolichols and dolichyl phosphate during chemical carcinogenesis in the rat by using the short-term assay systems of Ito et al. [21,22]. The dolichol contents in liver and liver microsomes of rats treated with diethylnitrosamine or 3’-methyl-4-dimethylaminoazobenzene as an initiator were approx. 1.5-times higher than those of the control rats and rats treated with only phenobarbital, though there was no change in the composition of dolichols in any of the groups. Hyperplastic nodules were generated in the liver with a high incidence when rats were treated with both the initiator and the promoter. Thus, the appearance of hyperplastic nodules did not correspond directly to the change in dolichol content. Dolichol monophosphatase activity and HMGCoA reductase (the major regulatory enzyme of cholesterol and dolichol biosynthesis) were also unchanged among the groups. The results indicate that the increase of dolichol is not due to changes in dolichol monophosphatase activity or HMGCoA reductase activity. At present, we can not explain the mechanism of the increase of the

573

dolichyl phosphate reacts with different sugars or whether there are differences in the extent of the reaction, the specificity or the ability of individual dolichyl phosphate homologs to accept sugars, but these results indicate that abnormal glycosylation may be involved in the process of formation of hyperplastic liver nodules. As a next step, it is necessary to measure the composition of dolichyl phosphate which participates in dolichol pyrophosphate oligosaccharide during the formation of hyperplastic liver nodules. It is noteworthy that the composition of dolichyl phosphate in the hyperplastic liver nodules was changed, but that of dolichol was not changed. This result indicates that the major part of dolichyl phosphate in the liver is synthesized de novo, as reported by Ekstrom et al. 1311 and Astrand et al. 1321. References 1 Butterworth,

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1,

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Days after hepatectomy

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%(days)

Fig. 6. Time courses of dolichol monophosphatase activity the regenerating liver microsomes. Conditions (and symbols) in Fig. 1A.

3

in as

dolichol content in the initiator-treated liver. However, this phenomenon might be related to the initiation process of chemical carcinogenesis in rat liver. On days 3, 14 and 35 after partial hepatectomy, the concentration of dolichyl phosphate in the liver of rats treated with both the initiator and the promoter was significantly higher than those in the other groups. Moreover, the composition of dofichyl phosphate in the rat liver treated with both the initiator and the promoter changed from day 7. The dependence of some glycosyltransferases on dolichyl phosphate of different chain lengths has been studied by Tosheva et al. [29] and Okamoto et al. [30]. They showed that there were differences in the function of some glycosyltransferases with dolichyl phosphate of different chain lengths between normal cells and hepatoma cells. We do not know whether the same

7 8 9 10 11 12 13

14 15 16 17

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