Glutathione and Gamma Glutamyl Transpeptidase in Rat Liver During Chemical Carcinogenesis" 2, 3 Silvio Fiala,4 Anish Mohindru,s William G. Kettering,4 Anna E. Fiala,s and Harold P. Morris 6 ,7
Our previous observations showed that the administration of several carcinogens led to a striking increase in the activity of y-glutamyl transpeptidase from very low starting values to activities up to 100 times as high in chemically induced, primary, and transplantable rat hepatomas. The effects of the carcinogens differed only in the rate at which GTase increased during their administration (1-5). The increasing GTase level was similar to the fetal or neonatal state in which the GTase level in rat liver was also high (3). GTase is integrated into the system of six enzymes of the y-glutamyl cycle, which has been credited with the physiologic role of the transfer of amino acids (via GSH) across the cell membrane (6). Within this system, GTase is credited with a translocation function and a carrier function is attributed to the tripeptide GSH (6). The activation of other enzymes of the y-glutamyl cycle during hepatocarcinogenesis is not known, but based on our studies, we believe that GTase is closely connected with the process of carcinogenesis in rat liver (2-5). Moreover, several factors indicate that GSH probably plays a role in chemical carcinogenesis in rat liver: 1) It is the in vivo effector of GTase. 2) After administration of hepatocarcinogens, GSH concentration in rat liver increases during the precancerous stage (7-11). 3) GSH occurs primarily in the soluble phase; part of it conjuVOL. 57, NO.3, SEPTEMBER 1976
gates with foreign compounds or their metabolites for detoxification and transport from the body (12). Similar reactions may occur with some carcinogens. 4) Another portion of GSH forms mixed disulfides with protein-SH groups (13, 14). This protein-bound GSH may be released after reduction of the disulfide bond by an exchange reaction with other thiols or by depletion of free GSH with which the protein-bound GSH may exist in a dynamic equilibrium. Free GSH protects the -SH groups from binding to radiation-induced radicals (13) or electrophilic drug metabolites (14). Depletion of the reservoir of free GSH enhanced both the covalent binding of toxic electrophilic metabolites to cell macromolecules and the degree of cell damage (14). The protection or sensitivity of target cells against many carcinogens may also depend on these reactions. Before we could explore this important problem,8 it was necessary to clarify the roles of GSH and GTase and their relationship during hepatocarcinogenesis. Since carcinogenic compounds can activate GTase and GSH, we administered compounds such as 3' -MeDAB to rats and then investigated the effects of various factors on GSH and GTase levels during treatment. We also correlated changes in GTase and GSH levels with gross pathologic changes during the administration of hepatocarcinogens in 12 transplantable Morris hepatomas. MATERIALS AND METHODS
Transplantable hepatomas, strains of hepatoma-bearing rats, preparation of tissue extracts, and carcinogen dosages were described earlier (4, 5). Animals.-Male Sprague-Dawley rats were used, unless specified otherwise. Normal, adrenalectomized, and hypophysectomized rats were purchased from Carworth Farms, Wilmington, Massachusetts. Compounds.-DAB, 2-Me-DAB, and 3'-Me-DAB were ABBREVIATIONS USED: GTase=gamma glutamyl transpeptidase; GSH=glutathione; SH=sulfhydryl; 3'-Me-DAB=3'-methyl-4-dimethylaminoazobenzene; DAB=4-dimethylaminoazobenzene; 2-MeDAB=2-methyl-4-dimethylaminoazobenzene; MCA=3-methylcholanthrene; 2-AAF=2-acetylaminofluorene; DMN = dimethylnitrosamine . I Submitted March 19, 1975; revised December 4, 1975; accepted February 20, 1976. 2 Supported by Veterans Administration project 2812-01 and Public Health Service grant CAI4084 from the National Cancer Institute. 3 Presented in part at the Annual Meeting of the American Society of Biological Chemists, Minneapolis, Minn., June 6,1974. 4 Laboratory of Cell Physiology , Veterans Administration Center, Martinsburg, W. Va. 25401. 5 Shepherd College, Shepherdstown, W. Va. 25443. 6 Department ofBiochemistry, Howard University College of Medicine, Washington, D.C. 20001. 7 We thank Mr. Henry Grantham of the Laboratory of Cell Physiology for preparing the tissues. 8 Fiala S, Trout EC Jr, Kettering WG: In preparation.
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ABSTRACT-Continued administration of several hepatocarcin· ogens led to an increase in the concentration of glutathion·e (GSH) in the livers of intact, but not of hypophysectomized or adrenalectomized rats. The concentration of GSH remained high until the development of hyperplastic nodules. Subsequently, the concentration of GSH dropped to the normal level or below. A single dose of 3'-methyl-4-dimethylaminoazobenzene (3'-MeDAB) produced an increase of GSH which, within a certain range, depended upon the amount of the carcinogen. In well differentiated, slowly growing hepatomas, the concentration of GSH approached the level in normal adult rat liver. On the other hand, in nondifferentiated and rapidly growing hepatomas, GSH was only 30-40% of that in normal liver. The activity of y-glutamyl transpeptidase (GTase) increased within 24-48 hours after a single large dose of 3'-Me-DAB. Continued feeding of 3'-Me-DAB led to an exponential increase of GTase. During hepatocarcinogenesis, the level of GTase activity corresponded to the degree and size of pathologic changes produced in rat liver. Chloramphenicol partially inhibited the increase of GTase induced by 2-acetylaminofluorene. Pretreatment with 3-methylcholanthrene partially inhibited the increase of GTase that had been induced by a single dose of3'-Me-DAB. Puromycin partially inhibited the increase of GTase induced by several doses of dimethylnitrosamine. These observations indicated a close connection between the activation of GTase a~d chemical carcinogenesis in rat liver. Measurements of GTase activity in 12 Morris hepatomas supported this conclusion; their GTase levels were greatly elevated compared with that in normal adult rat liver.-J Nall Cancer Inst 57: 591-598, 1976.
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FIALA ET AL.
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modification (4,5) of the Orlowski and Meister method (19). To prepare the substrate, a mixture of25 ml TrisHCl buffer (pH 7.6) and 73.8 mg N -glycylglycine was saturated with 31.6 mg L-y-glutamyl-p-nitroanilide and stirred at 54° C until clear. The residue was filtered off; the solution, kept in a darkened flask, was used within 2 hours. Since GTase in hepatomas is located in subcellular granules with practically no measurable activity in the soluble phase (4), buffer extracts, homogenates, and supernatants are unsuitable for GTase assays. To avoid losses, GTase assays were made with weighed amounts of acetone-powdered liver or hepatoma tissue (1-20 mg; 1 g powdered tissue=3.57 g tissue, wet wt). Measurements were expressed in units equal to nanomoles of pnitroaniline liberated by 1 g tissue (wet wt) in 1 minute under our standard laboratory conditions (30 min at 37° C, pH 7.6). DNA (1 U DNA=5xlO- 12 g) was determined by Schneider's method (20). RESULTS Levels of GSH and GTase During Chemical Carcinogenesis in Rat Liver
A single dose of at least 10 mg 3' -Me-DAB/100 g body weight, administered by stomach tube, increased the GSH level by approximately 60-100% within 48 hours. An equally high increase was produced by the same amounts of 2-AAF. DAB and 2-Me-DAB produced comparable effects. In contrast, the same concentration of chrysoidine produced no detectable increase of GSH (text-fig. 1). The elevated GSH levels returned to normal within 72-96 hours. Within this time, 2-Me-DAB and chrysoidine did not increase GTase activity detectably. DAB, 3'-Me-DAB and 2-AAF increased GTase activity by 25, 100, and 140%, respectively. In one instance, a single dose of 25 mg 3'-Me-DAB/l00 g increased the level of GTase by 460% in 72 hours, which coincided with a GSH level of about 80% above normal. To a certain extent, the individual dosage of 3' -MeDAB determined how much GSH increased in 24 hours (text-fig. 2), although we noted a lack of linearity. The saturation effect was evident; larger amounts of the azo dye did not produce a greater increase of GSH within 24 hours, though it did often increase after 48 hours. GSH measurements with Ellman's reaction gave similar results. The elevation of GSH during continued feeding with 3'-Me-DAB varied from day to day; it was usually highest during the first weeks, but it remained high until the development of hyperplastic nodules at 60-65 days (text-fig. 3). The effects of2-AAF were similar, but DAB and DL-ethionine produced smaller and slower increases in GSH concentration. Noncarcinogenic (or weakly carcinogenic) 2-Me-DAB produced only a small elevation of GSI-;I during the first 3 weeks. In contrast with the variation of GSH levels, the activity of GTase steadily increased during continued carcinogen feeding. Accumulation of GTase was dependent on the total dose (D) of 3'-Me-DAB, which was equal to the product of the average daily dose (d) of the carcinogen and the number of days, (t): D=dxt (21). The course of GTase activity during hepatocarcinogenesis VOL 57, NO.3, SEPTEMBER 1976
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synthesized in our laboratory. The water-soluble azo dye chrysoidine Y (2,4-diaminoazobenzene hydrochloride), MCA, 2-AAF, and DMN were purchased from Eastman Kodak Company, Rochester, New York. Puromycin was obtained from Calbiochem, La Jolla, California. Chloramphenicol was purchased from Sigma Chemical Company, St. Louis, Missouri. Tissue examination.-Periodically during carcinogen administration and hepatoma development, tissue specimens were fixed in 15% formaldehyde, embedded in paraffin, and stained with hematoxylin and eosin. Hyperplastic nodules (most of which were obvious) were identified according to histologic guidelines of Epstein et al. (15). GSH assay in tissue extracts.-Rats were killed by chloroform. A piece of liver was excised, and 500 mg was quickly weighed and homogenized at 0° C in a glass homogenizer with 2 ml of 15% metaphosphoric acid. After 3-5 minutes, three volumes of sodium phosphate buffer (0.2 M, pH 7.6) were added, which brought the pH from 1.0-1.5 to 6.0-6.5 and the volume to 8 ml. The extract was centrifuged at 15,000Xg for 10 minutes at 4° C. GSH was assayed by the "305" reaction (absorption spectra, with maxima at 305 nm) with alloxan (16) because of its specificity. A standard curve prepared with commercial GSH (Sigma Chemical Co., St. Louis, Mo.) was linear, with a range of 0-16 f-tg corresponding to the data of Patterson and Lazarow (16), but the results were not strictly linear when the 305 reaction was applied to tissue extracts. Our assay was a modification of the method of Suga et al. (17). The test contained 2.7 ml sodium phosphate buffer (0.2 M, pH 7.6),0.9 ml of 0.1 N NaOH, 0.4 ml extract, and 1.0 ml freshly made aqueous solution of 0.1 % alloxan (Eastman); in the control, alloxan was replaced by distilled water. The sample was allowed to stand for 10 minutes at room temperature before the spectrum was recorded at a range of 350-230 nm with a Beckman Acta III spectrophotometer. Since the acid content caused a drop of 0.1-0.2 in pH, we corrected the optical density measurements by 7% to correspond with optimal values at pH 7.6. Under these conditions the average GSH content was 234 mg/lOO g normal rat liver. This agreed with the work of Masayama et al. (7), who had obtained data by the iodometric method and subtracted the amounts of ascorbic acid. It also agreed with values we obtained by Ellman's nonspecific reaction (18) with the use of 5,5' -dithiobis-(2-nitrobenzoic acid). In these tests an extract from 0.5 g liver was prepared as described above, except that the sodium phosphate used for dilution was 0.1 M, pH 8.0. The supernatant (0.5 ml) was further diluted with 9.5 ml of the same buffer. Using 3-ml aliquots for this assay, we measured optical density at 412 nm and calculated the amount of -SH according to formula (18). We prevented postmortem decom position of GSH (by high levels of GTase) in guinea pig livers or rat hepatomas by quickly excising tissue specimens, freezing them in liquid nitrogen, weighing them on a torsion balance, and homogenizing them immediately in 15% metaphosphoric acid. GTase and DNA assays.-GTase was determined by our
593
GSH AND GTASE IN RAT LIVER CARCINOGENESIS 400
300
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I.-Levels of GSH in rat liver at 24 and 48 hours after a single oral dose of 2 ml corn oil (control), and 2 ml corn oil plus compound (10 mg/ 100 g body wt). Shown are absorption spectra with maxima at 305 nm obtained by the reaction with alloxan.
TEXT-FIGURE
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TEXT-FIGURE
3.-Effects of continued administration of 3' -Me-DAB
(8), 2-Me-DAB (0), DAB (e), and basal diet without azo dye (A) on
GSH levels in rat liver. Concentration of azo dyes was 0.06%.
produced by moderately carcinogenic DAB was similar but much slower. (7) 218.4±38.3 200+--,__, -____. -__________
o
5
10
20
40
mo 3-Me-DAB 2.-GSH levels in rat liver 24 hours after administration of single oral doses of various amounts of 3'-Me-DAB to 300-g male Sprague-Dawley rats. Values are meanS±SD.
TEXT-FIGURE
produced by 3'-Me-DAB was exponential, with the logarithm of GTase increase, In(At/An), a linear function of time (text-fig. 4). In this equation, At equals the GTase level at various times in 3'-Me-DAB-fed rats, and An equals GTase activity at various times in control rats. Other data indicated that the doubling time of GTase activity during 3' -Me-DAB-induced carcinogenesis was approximately 10 days. The course of GTase mcrease VOL. 57, No.3, SEPTEMBER 1976
Influence of Various Factors on GSH and GTase Levels During Hepatocarcinogen Administration
The data in table 1 indicate that hypophysectomy abolished the increase of GSH produced by a single large dose of 3'-Me-DAB. Twenty-four hours after administration of corn oil alone or with azo dye, the GSH level dropped considerably below the normal level. Adrenalectomy inhibited both the early decrease and later increase of GSH seen after the administration of 3' -MeDAB (table 1). Chloramphenicol, when administered with 2-AAF, markedly inhibited the upward trend ofGTase (text-fig. 5). In sharp contrast, it did not inhibit the increase of GSH induced by 2-AAF; in fact, the total increase was approximately the sum of GSH increases induced separately by the two compounds (text-fig. 6). Administered
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NATL CANCER INST,
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strongly inhibited, and two doses (30 and 15 mg) abolished, the increase of GTase induced by 3'-Me-DAB. Table 3 shows the elevation of GTase that was produced by DMN, with or without puromycin. We limited DMN injections to three, or at most, four, to avoid toxic effects and necrosis that could cause proteolysis and invalidate the results. The increase of GTase induced by three doses of DMN was inhibited but not abolished by puromycin. After four doses of DMN, puromycin inhibited the induction of GTase by approximately 50%, but the toxic effects of DMN were already apparent. The dependence on the formation of new RNA could not be established with actinomycin D.
TEXT-FIGURE 4.-Course of GTase increase during carcinogenesis in rat liver, expressed as In (AtlAn) , where At represents the GTase level at various times in 3'-Me-DAB-treated rats, and An is the activity in the liver of rats on basal diet at the same times.
1.7
TABLE
l.-GSH levels in rats after administration of a single large dose of 3 '-Me-DAB GSH concentrations" mg/100 g liver (Number of rats tested)
Substances administered orally to rats"
o min
4 hr
121
1.5
[GSHl, 1.4 iGSHJ, 1.3
24 hr L2
229±16.9 (13)
Normal Corn oil 3'-Me-DAB Hypophysectomized Corn oil 3 '-Me-DAB Adrenalectomized Corn oil 3'-Me-DAB
212.7 ± 15.7 (4) 216.2±41.3 (6) 198.2± 5.5 (3) 331.l±19.1 (4) 257±14.0
(3) 244.2±14.1 (3) 198.9±20.6 (3) 235.3±18.3 (4) 199.1±25.5 (6)
190.2±7.9
(4) 205.2±20.3 (2) 197.7±56.1 (3) 193.8± 7.9 (2) 163.6±54.8 (4)
" Animals killed for testing at 0 min were not fed. Controls received 2 ml corn oil; 10 mg 3' -Me-DAB/2 ml corn oil was administered to the other rats. b Values are meanS±SD.
28
21 Days on Diet
14
7
35
TEXT-FIGURE 6.-Increase of GSH concentration in the livers of rats fed 2-AAF (6), chloramphenicol (0), and 2-AAF plus chloramphenicol (e) in basal diet for 35 days. GSH increase is expressed as [GSH)tI[GSH]o, where [GSH)t represents GSH concentration in 100 g liver from rats fed 1-35 days (f), and [GSH)o in 100 g liver was 234±8 mg.
600
2.-Effects of selected dosages of MeA upon 3'-Me-DAB-induced activation of GSH and GTase
500
Effects on GSH and GTase activation (Number of rats tested)" CZI
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Substances administered orally to rats"
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121
GTase GSH mg/100 g
Ur
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10
20
30
40
Days on Diet
TEXT-FIGURE 5.-Comparative levels of GTase in rats fed basal diet (6), basal diet plus 0.04% 2-AAF (8), and basal diet plus 2% chloramphenicol and 0.04% 2-AAF (e) for 35 days. Nofe inhibition by chloramphenicol of 2-AAF-induced activation of GTase.
alone at a 2% concentration in basal diet, chloramphenicol caused a gradual increase of GSH, which peaked after 3 weeks (text-fig. 6). Table 2 shows that a single large dose of MeA J NATL CANCER INST
Corn oil MCA 3 '-Me-DAB MCA, 1 dose +3'-Me-DAB MCA, 2 doses +3 '-Me-DAB
40 hr 40 hr 40;46 hr 40 hr 46 hr
234±12 281±53 376±9 299±5 383
(7) 75±7 (3) 66±2 (2) 120±20 (2) 90±10 68
Percent increase
Percent inhibition
60 20
66 100
" Control animals received 2 ml corn oil. MCA was administered in 2 ml corn oil (1 dose=30 mg; 2 doses=30 mg, then 15 mg 25 hr later). 3'-Me-DAB (10 mg/2 ml corn oil) was administered alone or with the first dose of MCA. GSH and GTase assays were performed 40 hr after a single dose of MCA; assays were performed 46 hr after two doses of MCA. b Values are meanS±SD. U =nanomoles of p-nitroaniline liberated by 1 g tissue in 1 min, under our standard laboratory conditions (30 min at 37° C, pH 7.6). C
VOL 57, No.3, SEPTEMBER 1976
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121
1.6
595
GSH AND GTASE IN RAT LIVER CARCINOGENESIS
Correlation Between GTase and GSH Levels and Gross Pathologic Changes in Rat Liver During Chemical Carcinogenesis
Livers of animals that had been fed 3'-Me-DAB for 45 weeks had slightly granular surfaces and higher levels of GTase than did livers of control animals. The areas with scattered hyperplastic nodules (confirmed histologically) had considerably higher GTase activity than did tissues without hyperplastic nodules. GTase activity in primary hepatomas exceeded the level of GTase in any precancerous liver tissues. All our observations indicated that the level of GTase during 3'-Me-DAB-induced carcinogenesis approximated the accumulation of macroscopic and histologic pathologic changes in rat liver. Typical examples are illustrated in table 4. The data show that gross macroscopic changes were accompanied by the appearance of different and smaller cells, as manifested by larger amounts of DNA/unit weight in pathologically changed areas of tissue, compared with relatively normal adjacent areas. TABLE
3.-Inhibition by puromycin of DMN-induced activation ofGTase Effects of DMN and puromycin on GTase levels b (Number of rats tested) Four injections DMN
Three injections DMN Substances administered to rats· GTase activity U Physiologic saline DMN DMN+puromycin Puromycin
50±11 100±8 71±2 63±4
Percent increase after DMN injections
(4) (4) (4) (2)
Percent inhibition after puromycin injections
100 42-26=16 26
GTase activity U 50±11 180±50 137 :t31 66
84
(4) (3) (3) (1)
Percent increase after DMN injections
Percent inhibition after puromycin injections
260 174-32=142 32
45
• DMN dose, 6 mg/injection=15 mg/kg body weight; puromycin dose, 10 mg/injection. Values expressed are meanS±SD.
b
TABLE
4.-Correlations between pathologic changes, GSH concentration, and GTase activity during chemical carcinogenesis in rat livers Comparisons of GSH, GTase, and DNA measurements during carcinogenesis
Diets of typical rats; stages oflesion development·
Normal food (control) No lesions 3'-Me-DAB, 61 days; and normal food, 15 days No lesions Granular surfaces Hyperplastic nodules 3'-Me-DAB, 76 days; and normal food, 26 days Primary hepatomas DAB, 144 days; and normal food, 18 days Mixed hepatomas Primary hepatomas
A GSH mg/l00 g tissue
B GTase activity U/g tissue
C DNA U/(g tissue X 106)
Db
EC
Fd
GSH, pg DNA,U
GTase UDNA X 10-6
GTase, U GSH,mg
231
75
400
5.77
0.19
32
243 247 267
229 423 5,355
429 488 976
5.66 5.06 2.73
0.53 0.87 5.49
94 171 2,005
172
7,500
1,176
1.46
6.38
4,360
239 165
6,383 10,883
688 1,200
3.47 1.38
9.28 9.07
2,671 6,596
Azo dye concentration was 0.06% in basal diet. 10xcolumn A b Column D measurements = column C column B CColumn E measurements = column C . 100xcolumn B d Column F measurements "" column A a
VOL. 57, NO.3, SEPTEMBER 1976
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Noncarcinogenic 2-Me-DAB did not produce any permanent increase of GTase activity during 150 days of feeding (except a small transitory increase during the first 2-3 wk). Text-figure 3 indicates that GSH remained elevated for several weeks of 3'-Me-DAB feeding, while GTase increased steadily. For the first 50 days, the GSH:DNA ratio was elevated and the GTase:DNA ratio increased. As we showed previously (22), the amount of DNA remained low until massive cell proliferation occurred in the rat liver. With the onset of massive cell proliferation, manifested by an increase of total DNA (easily measured by Schneider's method) that coincided with the appearance of hyperplastic nodules, the GSH concentration (based on units of DN A) in these areas decreased considerably. The GSH level was strikingly decreased at the late stage of hepatocarcinogenesis in 3'-Me-DAB-induced primary hepatomas and rapidly growing, nondifferentiated hepatomas, e.g., N ovikoff and Yoshida hepato-
596
FIALA ET AL.
GTase Activity in Morris Hepatomas
We have seen no exception to high GTase activity in various chemically-induced rat hepatomas (5), although in a few instances we could not ascertain to what extent the variations in GTase activity might have been influenced by the rates of growth, degrees of differentiation, and histologic properties of the hepatomas. If GTase activity during hepatocarcinogenesis corresponds approximately to the accumulation of macroscopic histologic changes, the variations among hepatomas (relative to differentiation and rate of growth) probably result in different GTase activities. Table 6 shows GTase levels in
12 transplantable hepatomas developed by Morris (23), in order of decreasing time spans between transfers. Correlation is poor between rate of growth and GTase activity. With the exception of hepatoma 7288c+c, the 2 most slowly growing hepatomas (which seemed to be the most differentiated) had the lowest GTase activity. Similar low GTase values were observed earlier (5) in a rapidly growing (4-5 days) ascites Novikoff hepatoma. In hepatoma 44, the low GTase activity/U weight cannot be ascribed simply to highly differentiated tumor cells, but more to the fact that a large part of this tumor consisted of connective tissue with islands of large hepatocyte-like tumor cells. TABLE 6.-GTase activity in Morris hepatomas Hepatoma (Number of tumors) 44 (2) 9121 F (2) RI 9618 A2 R3B 7288 c+c 3924 A 9618 A2 7794 B 7288 C 5123 B H 35+c (2) 8994 B (2)
Generation
Days between transfers
18 77 74 90 46 32 337 91 62 80 168 258 140
161 70 70 49 44 35 31 31 28 21 20 16 14
GTase activity U
Degree of differentiation'
361-645 1,151-1,484 2,902 3,684 3,940 2,562 3,963 3,967 5,666 4,959 4,936 4,902-11,610 2,583-3,745
H W R P N R N N P N P P N
• The following distinctions describe the degree to which hepatomas were differentiated: H=highly; W = well; R=relatively well; P=poorly; N =nondifferentiated.
TABLE 5.-Correlations between levels of GSH and GTase and the degrees of differentiation in rat livers and hepatomas GSH, GTase, and DNA measurements in rat livers and hepatomas (Number of rats tested) Tissue
Liver Fetal Neonatal Adult Hepatomas Primary H-207 Yoshida Morris 9121F 5123+c 8994
441
Age'
Degree of differentiation in hepatomas b
-3 days 18 hr 3 mo 8 wk 6-7 days 10 wk 4 wk 14 days 7 mo
W W
N W
P N W
E GTase UDNA x 1O-6d
GTase e GSH
2,790 (3) 3,759 (3) 75 (20)
2.63 4.61 0.19
2,536 2,685 32
4.64 3.73 0.87
7,564 (1) 7,500 (12) 4,950 (4)
15.0 15.12 3.10
3,232 4,054 3,536
3.78 2.36 0.61 1.32
1,728 6,921 3,675 423
2.28 6.19 3.22 0.51
604 2,622 5,250 384
D
A DNA in g tissue Ux10 6
B GSH mg/100 g tissue
C GSH pg/U DNAc
GTase U/g tissue
1,060 816 400
110 (2) 140 (2) 234 (20)
1.04 1.72 5.85
504 496 1,600
234 (1) 185 (8) 140 (4)
756 1,118 1,142 832
286 264 70 110
(2) (2) (2) (2)
F
• Primary hepatoma was taken from a rat that had been fed DAB for 114 days and normal food for 213 days. b For liver tissue differentiation, fetal was
Our previous observations showed that the administration of several carcinogens led to a striking increase in the activity of y-glutamyl transpeptidase from very low starting values to activities up to 100 times as high in chemically induced, primary, and transplantable rat hepatomas. The effects of the carcinogens differed only in the rate at which GTase increased during their administration (1-5). The increasing GTase level was similar to the fetal or neonatal state in which the GTase level in rat liver was also high (3). GTase is integrated into the system of six enzymes of the y-glutamyl cycle, which has been credited with the physiologic role of the transfer of amino acids (via GSH) across the cell membrane (6). Within this system, GTase is credited with a translocation function and a carrier function is attributed to the tripeptide GSH (6). The activation of other enzymes of the y-glutamyl cycle during hepatocarcinogenesis is not known, but based on our studies, we believe that GTase is closely connected with the process of carcinogenesis in rat liver (2-5). Moreover, several factors indicate that GSH probably plays a role in chemical carcinogenesis in rat liver: 1) It is the in vivo effector of GTase. 2) After administration of hepatocarcinogens, GSH concentration in rat liver increases during the precancerous stage (7-11). 3) GSH occurs primarily in the soluble phase; part of it conjuVOL. 57, NO.3, SEPTEMBER 1976
gates with foreign compounds or their metabolites for detoxification and transport from the body (12). Similar reactions may occur with some carcinogens. 4) Another portion of GSH forms mixed disulfides with protein-SH groups (13, 14). This protein-bound GSH may be released after reduction of the disulfide bond by an exchange reaction with other thiols or by depletion of free GSH with which the protein-bound GSH may exist in a dynamic equilibrium. Free GSH protects the -SH groups from binding to radiation-induced radicals (13) or electrophilic drug metabolites (14). Depletion of the reservoir of free GSH enhanced both the covalent binding of toxic electrophilic metabolites to cell macromolecules and the degree of cell damage (14). The protection or sensitivity of target cells against many carcinogens may also depend on these reactions. Before we could explore this important problem,8 it was necessary to clarify the roles of GSH and GTase and their relationship during hepatocarcinogenesis. Since carcinogenic compounds can activate GTase and GSH, we administered compounds such as 3' -MeDAB to rats and then investigated the effects of various factors on GSH and GTase levels during treatment. We also correlated changes in GTase and GSH levels with gross pathologic changes during the administration of hepatocarcinogens in 12 transplantable Morris hepatomas. MATERIALS AND METHODS
Transplantable hepatomas, strains of hepatoma-bearing rats, preparation of tissue extracts, and carcinogen dosages were described earlier (4, 5). Animals.-Male Sprague-Dawley rats were used, unless specified otherwise. Normal, adrenalectomized, and hypophysectomized rats were purchased from Carworth Farms, Wilmington, Massachusetts. Compounds.-DAB, 2-Me-DAB, and 3'-Me-DAB were ABBREVIATIONS USED: GTase=gamma glutamyl transpeptidase; GSH=glutathione; SH=sulfhydryl; 3'-Me-DAB=3'-methyl-4-dimethylaminoazobenzene; DAB=4-dimethylaminoazobenzene; 2-MeDAB=2-methyl-4-dimethylaminoazobenzene; MCA=3-methylcholanthrene; 2-AAF=2-acetylaminofluorene; DMN = dimethylnitrosamine . I Submitted March 19, 1975; revised December 4, 1975; accepted February 20, 1976. 2 Supported by Veterans Administration project 2812-01 and Public Health Service grant CAI4084 from the National Cancer Institute. 3 Presented in part at the Annual Meeting of the American Society of Biological Chemists, Minneapolis, Minn., June 6,1974. 4 Laboratory of Cell Physiology , Veterans Administration Center, Martinsburg, W. Va. 25401. 5 Shepherd College, Shepherdstown, W. Va. 25443. 6 Department ofBiochemistry, Howard University College of Medicine, Washington, D.C. 20001. 7 We thank Mr. Henry Grantham of the Laboratory of Cell Physiology for preparing the tissues. 8 Fiala S, Trout EC Jr, Kettering WG: In preparation.
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ABSTRACT-Continued administration of several hepatocarcin· ogens led to an increase in the concentration of glutathion·e (GSH) in the livers of intact, but not of hypophysectomized or adrenalectomized rats. The concentration of GSH remained high until the development of hyperplastic nodules. Subsequently, the concentration of GSH dropped to the normal level or below. A single dose of 3'-methyl-4-dimethylaminoazobenzene (3'-MeDAB) produced an increase of GSH which, within a certain range, depended upon the amount of the carcinogen. In well differentiated, slowly growing hepatomas, the concentration of GSH approached the level in normal adult rat liver. On the other hand, in nondifferentiated and rapidly growing hepatomas, GSH was only 30-40% of that in normal liver. The activity of y-glutamyl transpeptidase (GTase) increased within 24-48 hours after a single large dose of 3'-Me-DAB. Continued feeding of 3'-Me-DAB led to an exponential increase of GTase. During hepatocarcinogenesis, the level of GTase activity corresponded to the degree and size of pathologic changes produced in rat liver. Chloramphenicol partially inhibited the increase of GTase induced by 2-acetylaminofluorene. Pretreatment with 3-methylcholanthrene partially inhibited the increase of GTase that had been induced by a single dose of3'-Me-DAB. Puromycin partially inhibited the increase of GTase induced by several doses of dimethylnitrosamine. These observations indicated a close connection between the activation of GTase a~d chemical carcinogenesis in rat liver. Measurements of GTase activity in 12 Morris hepatomas supported this conclusion; their GTase levels were greatly elevated compared with that in normal adult rat liver.-J Nall Cancer Inst 57: 591-598, 1976.
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FIALA ET AL.
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modification (4,5) of the Orlowski and Meister method (19). To prepare the substrate, a mixture of25 ml TrisHCl buffer (pH 7.6) and 73.8 mg N -glycylglycine was saturated with 31.6 mg L-y-glutamyl-p-nitroanilide and stirred at 54° C until clear. The residue was filtered off; the solution, kept in a darkened flask, was used within 2 hours. Since GTase in hepatomas is located in subcellular granules with practically no measurable activity in the soluble phase (4), buffer extracts, homogenates, and supernatants are unsuitable for GTase assays. To avoid losses, GTase assays were made with weighed amounts of acetone-powdered liver or hepatoma tissue (1-20 mg; 1 g powdered tissue=3.57 g tissue, wet wt). Measurements were expressed in units equal to nanomoles of pnitroaniline liberated by 1 g tissue (wet wt) in 1 minute under our standard laboratory conditions (30 min at 37° C, pH 7.6). DNA (1 U DNA=5xlO- 12 g) was determined by Schneider's method (20). RESULTS Levels of GSH and GTase During Chemical Carcinogenesis in Rat Liver
A single dose of at least 10 mg 3' -Me-DAB/100 g body weight, administered by stomach tube, increased the GSH level by approximately 60-100% within 48 hours. An equally high increase was produced by the same amounts of 2-AAF. DAB and 2-Me-DAB produced comparable effects. In contrast, the same concentration of chrysoidine produced no detectable increase of GSH (text-fig. 1). The elevated GSH levels returned to normal within 72-96 hours. Within this time, 2-Me-DAB and chrysoidine did not increase GTase activity detectably. DAB, 3'-Me-DAB and 2-AAF increased GTase activity by 25, 100, and 140%, respectively. In one instance, a single dose of 25 mg 3'-Me-DAB/l00 g increased the level of GTase by 460% in 72 hours, which coincided with a GSH level of about 80% above normal. To a certain extent, the individual dosage of 3' -MeDAB determined how much GSH increased in 24 hours (text-fig. 2), although we noted a lack of linearity. The saturation effect was evident; larger amounts of the azo dye did not produce a greater increase of GSH within 24 hours, though it did often increase after 48 hours. GSH measurements with Ellman's reaction gave similar results. The elevation of GSH during continued feeding with 3'-Me-DAB varied from day to day; it was usually highest during the first weeks, but it remained high until the development of hyperplastic nodules at 60-65 days (text-fig. 3). The effects of2-AAF were similar, but DAB and DL-ethionine produced smaller and slower increases in GSH concentration. Noncarcinogenic (or weakly carcinogenic) 2-Me-DAB produced only a small elevation of GSI-;I during the first 3 weeks. In contrast with the variation of GSH levels, the activity of GTase steadily increased during continued carcinogen feeding. Accumulation of GTase was dependent on the total dose (D) of 3'-Me-DAB, which was equal to the product of the average daily dose (d) of the carcinogen and the number of days, (t): D=dxt (21). The course of GTase activity during hepatocarcinogenesis VOL 57, NO.3, SEPTEMBER 1976
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synthesized in our laboratory. The water-soluble azo dye chrysoidine Y (2,4-diaminoazobenzene hydrochloride), MCA, 2-AAF, and DMN were purchased from Eastman Kodak Company, Rochester, New York. Puromycin was obtained from Calbiochem, La Jolla, California. Chloramphenicol was purchased from Sigma Chemical Company, St. Louis, Missouri. Tissue examination.-Periodically during carcinogen administration and hepatoma development, tissue specimens were fixed in 15% formaldehyde, embedded in paraffin, and stained with hematoxylin and eosin. Hyperplastic nodules (most of which were obvious) were identified according to histologic guidelines of Epstein et al. (15). GSH assay in tissue extracts.-Rats were killed by chloroform. A piece of liver was excised, and 500 mg was quickly weighed and homogenized at 0° C in a glass homogenizer with 2 ml of 15% metaphosphoric acid. After 3-5 minutes, three volumes of sodium phosphate buffer (0.2 M, pH 7.6) were added, which brought the pH from 1.0-1.5 to 6.0-6.5 and the volume to 8 ml. The extract was centrifuged at 15,000Xg for 10 minutes at 4° C. GSH was assayed by the "305" reaction (absorption spectra, with maxima at 305 nm) with alloxan (16) because of its specificity. A standard curve prepared with commercial GSH (Sigma Chemical Co., St. Louis, Mo.) was linear, with a range of 0-16 f-tg corresponding to the data of Patterson and Lazarow (16), but the results were not strictly linear when the 305 reaction was applied to tissue extracts. Our assay was a modification of the method of Suga et al. (17). The test contained 2.7 ml sodium phosphate buffer (0.2 M, pH 7.6),0.9 ml of 0.1 N NaOH, 0.4 ml extract, and 1.0 ml freshly made aqueous solution of 0.1 % alloxan (Eastman); in the control, alloxan was replaced by distilled water. The sample was allowed to stand for 10 minutes at room temperature before the spectrum was recorded at a range of 350-230 nm with a Beckman Acta III spectrophotometer. Since the acid content caused a drop of 0.1-0.2 in pH, we corrected the optical density measurements by 7% to correspond with optimal values at pH 7.6. Under these conditions the average GSH content was 234 mg/lOO g normal rat liver. This agreed with the work of Masayama et al. (7), who had obtained data by the iodometric method and subtracted the amounts of ascorbic acid. It also agreed with values we obtained by Ellman's nonspecific reaction (18) with the use of 5,5' -dithiobis-(2-nitrobenzoic acid). In these tests an extract from 0.5 g liver was prepared as described above, except that the sodium phosphate used for dilution was 0.1 M, pH 8.0. The supernatant (0.5 ml) was further diluted with 9.5 ml of the same buffer. Using 3-ml aliquots for this assay, we measured optical density at 412 nm and calculated the amount of -SH according to formula (18). We prevented postmortem decom position of GSH (by high levels of GTase) in guinea pig livers or rat hepatomas by quickly excising tissue specimens, freezing them in liquid nitrogen, weighing them on a torsion balance, and homogenizing them immediately in 15% metaphosphoric acid. GTase and DNA assays.-GTase was determined by our
593
GSH AND GTASE IN RAT LIVER CARCINOGENESIS 400
300
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I.-Levels of GSH in rat liver at 24 and 48 hours after a single oral dose of 2 ml corn oil (control), and 2 ml corn oil plus compound (10 mg/ 100 g body wt). Shown are absorption spectra with maxima at 305 nm obtained by the reaction with alloxan.
TEXT-FIGURE
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TEXT-FIGURE
3.-Effects of continued administration of 3' -Me-DAB
(8), 2-Me-DAB (0), DAB (e), and basal diet without azo dye (A) on
GSH levels in rat liver. Concentration of azo dyes was 0.06%.
produced by moderately carcinogenic DAB was similar but much slower. (7) 218.4±38.3 200+--,__, -____. -__________
o
5
10
20
40
mo 3-Me-DAB 2.-GSH levels in rat liver 24 hours after administration of single oral doses of various amounts of 3'-Me-DAB to 300-g male Sprague-Dawley rats. Values are meanS±SD.
TEXT-FIGURE
produced by 3'-Me-DAB was exponential, with the logarithm of GTase increase, In(At/An), a linear function of time (text-fig. 4). In this equation, At equals the GTase level at various times in 3'-Me-DAB-fed rats, and An equals GTase activity at various times in control rats. Other data indicated that the doubling time of GTase activity during 3' -Me-DAB-induced carcinogenesis was approximately 10 days. The course of GTase mcrease VOL. 57, No.3, SEPTEMBER 1976
Influence of Various Factors on GSH and GTase Levels During Hepatocarcinogen Administration
The data in table 1 indicate that hypophysectomy abolished the increase of GSH produced by a single large dose of 3'-Me-DAB. Twenty-four hours after administration of corn oil alone or with azo dye, the GSH level dropped considerably below the normal level. Adrenalectomy inhibited both the early decrease and later increase of GSH seen after the administration of 3' -MeDAB (table 1). Chloramphenicol, when administered with 2-AAF, markedly inhibited the upward trend ofGTase (text-fig. 5). In sharp contrast, it did not inhibit the increase of GSH induced by 2-AAF; in fact, the total increase was approximately the sum of GSH increases induced separately by the two compounds (text-fig. 6). Administered
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NATL CANCER INST,
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strongly inhibited, and two doses (30 and 15 mg) abolished, the increase of GTase induced by 3'-Me-DAB. Table 3 shows the elevation of GTase that was produced by DMN, with or without puromycin. We limited DMN injections to three, or at most, four, to avoid toxic effects and necrosis that could cause proteolysis and invalidate the results. The increase of GTase induced by three doses of DMN was inhibited but not abolished by puromycin. After four doses of DMN, puromycin inhibited the induction of GTase by approximately 50%, but the toxic effects of DMN were already apparent. The dependence on the formation of new RNA could not be established with actinomycin D.
TEXT-FIGURE 4.-Course of GTase increase during carcinogenesis in rat liver, expressed as In (AtlAn) , where At represents the GTase level at various times in 3'-Me-DAB-treated rats, and An is the activity in the liver of rats on basal diet at the same times.
1.7
TABLE
l.-GSH levels in rats after administration of a single large dose of 3 '-Me-DAB GSH concentrations" mg/100 g liver (Number of rats tested)
Substances administered orally to rats"
o min
4 hr
121
1.5
[GSHl, 1.4 iGSHJ, 1.3
24 hr L2
229±16.9 (13)
Normal Corn oil 3'-Me-DAB Hypophysectomized Corn oil 3 '-Me-DAB Adrenalectomized Corn oil 3'-Me-DAB
212.7 ± 15.7 (4) 216.2±41.3 (6) 198.2± 5.5 (3) 331.l±19.1 (4) 257±14.0
(3) 244.2±14.1 (3) 198.9±20.6 (3) 235.3±18.3 (4) 199.1±25.5 (6)
190.2±7.9
(4) 205.2±20.3 (2) 197.7±56.1 (3) 193.8± 7.9 (2) 163.6±54.8 (4)
" Animals killed for testing at 0 min were not fed. Controls received 2 ml corn oil; 10 mg 3' -Me-DAB/2 ml corn oil was administered to the other rats. b Values are meanS±SD.
28
21 Days on Diet
14
7
35
TEXT-FIGURE 6.-Increase of GSH concentration in the livers of rats fed 2-AAF (6), chloramphenicol (0), and 2-AAF plus chloramphenicol (e) in basal diet for 35 days. GSH increase is expressed as [GSH)tI[GSH]o, where [GSH)t represents GSH concentration in 100 g liver from rats fed 1-35 days (f), and [GSH)o in 100 g liver was 234±8 mg.
600
2.-Effects of selected dosages of MeA upon 3'-Me-DAB-induced activation of GSH and GTase
500
Effects on GSH and GTase activation (Number of rats tested)" CZI
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Substances administered orally to rats"
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121
GTase GSH mg/100 g
Ur
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10
20
30
40
Days on Diet
TEXT-FIGURE 5.-Comparative levels of GTase in rats fed basal diet (6), basal diet plus 0.04% 2-AAF (8), and basal diet plus 2% chloramphenicol and 0.04% 2-AAF (e) for 35 days. Nofe inhibition by chloramphenicol of 2-AAF-induced activation of GTase.
alone at a 2% concentration in basal diet, chloramphenicol caused a gradual increase of GSH, which peaked after 3 weeks (text-fig. 6). Table 2 shows that a single large dose of MeA J NATL CANCER INST
Corn oil MCA 3 '-Me-DAB MCA, 1 dose +3'-Me-DAB MCA, 2 doses +3 '-Me-DAB
40 hr 40 hr 40;46 hr 40 hr 46 hr
234±12 281±53 376±9 299±5 383
(7) 75±7 (3) 66±2 (2) 120±20 (2) 90±10 68
Percent increase
Percent inhibition
60 20
66 100
" Control animals received 2 ml corn oil. MCA was administered in 2 ml corn oil (1 dose=30 mg; 2 doses=30 mg, then 15 mg 25 hr later). 3'-Me-DAB (10 mg/2 ml corn oil) was administered alone or with the first dose of MCA. GSH and GTase assays were performed 40 hr after a single dose of MCA; assays were performed 46 hr after two doses of MCA. b Values are meanS±SD. U =nanomoles of p-nitroaniline liberated by 1 g tissue in 1 min, under our standard laboratory conditions (30 min at 37° C, pH 7.6). C
VOL 57, No.3, SEPTEMBER 1976
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121
1.6
595
GSH AND GTASE IN RAT LIVER CARCINOGENESIS
Correlation Between GTase and GSH Levels and Gross Pathologic Changes in Rat Liver During Chemical Carcinogenesis
Livers of animals that had been fed 3'-Me-DAB for 45 weeks had slightly granular surfaces and higher levels of GTase than did livers of control animals. The areas with scattered hyperplastic nodules (confirmed histologically) had considerably higher GTase activity than did tissues without hyperplastic nodules. GTase activity in primary hepatomas exceeded the level of GTase in any precancerous liver tissues. All our observations indicated that the level of GTase during 3'-Me-DAB-induced carcinogenesis approximated the accumulation of macroscopic and histologic pathologic changes in rat liver. Typical examples are illustrated in table 4. The data show that gross macroscopic changes were accompanied by the appearance of different and smaller cells, as manifested by larger amounts of DNA/unit weight in pathologically changed areas of tissue, compared with relatively normal adjacent areas. TABLE
3.-Inhibition by puromycin of DMN-induced activation ofGTase Effects of DMN and puromycin on GTase levels b (Number of rats tested) Four injections DMN
Three injections DMN Substances administered to rats· GTase activity U Physiologic saline DMN DMN+puromycin Puromycin
50±11 100±8 71±2 63±4
Percent increase after DMN injections
(4) (4) (4) (2)
Percent inhibition after puromycin injections
100 42-26=16 26
GTase activity U 50±11 180±50 137 :t31 66
84
(4) (3) (3) (1)
Percent increase after DMN injections
Percent inhibition after puromycin injections
260 174-32=142 32
45
• DMN dose, 6 mg/injection=15 mg/kg body weight; puromycin dose, 10 mg/injection. Values expressed are meanS±SD.
b
TABLE
4.-Correlations between pathologic changes, GSH concentration, and GTase activity during chemical carcinogenesis in rat livers Comparisons of GSH, GTase, and DNA measurements during carcinogenesis
Diets of typical rats; stages oflesion development·
Normal food (control) No lesions 3'-Me-DAB, 61 days; and normal food, 15 days No lesions Granular surfaces Hyperplastic nodules 3'-Me-DAB, 76 days; and normal food, 26 days Primary hepatomas DAB, 144 days; and normal food, 18 days Mixed hepatomas Primary hepatomas
A GSH mg/l00 g tissue
B GTase activity U/g tissue
C DNA U/(g tissue X 106)
Db
EC
Fd
GSH, pg DNA,U
GTase UDNA X 10-6
GTase, U GSH,mg
231
75
400
5.77
0.19
32
243 247 267
229 423 5,355
429 488 976
5.66 5.06 2.73
0.53 0.87 5.49
94 171 2,005
172
7,500
1,176
1.46
6.38
4,360
239 165
6,383 10,883
688 1,200
3.47 1.38
9.28 9.07
2,671 6,596
Azo dye concentration was 0.06% in basal diet. 10xcolumn A b Column D measurements = column C column B CColumn E measurements = column C . 100xcolumn B d Column F measurements "" column A a
VOL. 57, NO.3, SEPTEMBER 1976
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Noncarcinogenic 2-Me-DAB did not produce any permanent increase of GTase activity during 150 days of feeding (except a small transitory increase during the first 2-3 wk). Text-figure 3 indicates that GSH remained elevated for several weeks of 3'-Me-DAB feeding, while GTase increased steadily. For the first 50 days, the GSH:DNA ratio was elevated and the GTase:DNA ratio increased. As we showed previously (22), the amount of DNA remained low until massive cell proliferation occurred in the rat liver. With the onset of massive cell proliferation, manifested by an increase of total DNA (easily measured by Schneider's method) that coincided with the appearance of hyperplastic nodules, the GSH concentration (based on units of DN A) in these areas decreased considerably. The GSH level was strikingly decreased at the late stage of hepatocarcinogenesis in 3'-Me-DAB-induced primary hepatomas and rapidly growing, nondifferentiated hepatomas, e.g., N ovikoff and Yoshida hepato-
596
FIALA ET AL.
GTase Activity in Morris Hepatomas
We have seen no exception to high GTase activity in various chemically-induced rat hepatomas (5), although in a few instances we could not ascertain to what extent the variations in GTase activity might have been influenced by the rates of growth, degrees of differentiation, and histologic properties of the hepatomas. If GTase activity during hepatocarcinogenesis corresponds approximately to the accumulation of macroscopic histologic changes, the variations among hepatomas (relative to differentiation and rate of growth) probably result in different GTase activities. Table 6 shows GTase levels in
12 transplantable hepatomas developed by Morris (23), in order of decreasing time spans between transfers. Correlation is poor between rate of growth and GTase activity. With the exception of hepatoma 7288c+c, the 2 most slowly growing hepatomas (which seemed to be the most differentiated) had the lowest GTase activity. Similar low GTase values were observed earlier (5) in a rapidly growing (4-5 days) ascites Novikoff hepatoma. In hepatoma 44, the low GTase activity/U weight cannot be ascribed simply to highly differentiated tumor cells, but more to the fact that a large part of this tumor consisted of connective tissue with islands of large hepatocyte-like tumor cells. TABLE 6.-GTase activity in Morris hepatomas Hepatoma (Number of tumors) 44 (2) 9121 F (2) RI 9618 A2 R3B 7288 c+c 3924 A 9618 A2 7794 B 7288 C 5123 B H 35+c (2) 8994 B (2)
Generation
Days between transfers
18 77 74 90 46 32 337 91 62 80 168 258 140
161 70 70 49 44 35 31 31 28 21 20 16 14
GTase activity U
Degree of differentiation'
361-645 1,151-1,484 2,902 3,684 3,940 2,562 3,963 3,967 5,666 4,959 4,936 4,902-11,610 2,583-3,745
H W R P N R N N P N P P N
• The following distinctions describe the degree to which hepatomas were differentiated: H=highly; W = well; R=relatively well; P=poorly; N =nondifferentiated.
TABLE 5.-Correlations between levels of GSH and GTase and the degrees of differentiation in rat livers and hepatomas GSH, GTase, and DNA measurements in rat livers and hepatomas (Number of rats tested) Tissue
Liver Fetal Neonatal Adult Hepatomas Primary H-207 Yoshida Morris 9121F 5123+c 8994
441
Age'
Degree of differentiation in hepatomas b
-3 days 18 hr 3 mo 8 wk 6-7 days 10 wk 4 wk 14 days 7 mo
W W
N W
P N W
E GTase UDNA x 1O-6d
GTase e GSH
2,790 (3) 3,759 (3) 75 (20)
2.63 4.61 0.19
2,536 2,685 32
4.64 3.73 0.87
7,564 (1) 7,500 (12) 4,950 (4)
15.0 15.12 3.10
3,232 4,054 3,536
3.78 2.36 0.61 1.32
1,728 6,921 3,675 423
2.28 6.19 3.22 0.51
604 2,622 5,250 384
D
A DNA in g tissue Ux10 6
B GSH mg/100 g tissue
C GSH pg/U DNAc
GTase U/g tissue
1,060 816 400
110 (2) 140 (2) 234 (20)
1.04 1.72 5.85
504 496 1,600
234 (1) 185 (8) 140 (4)
756 1,118 1,142 832
286 264 70 110
(2) (2) (2) (2)
F
• Primary hepatoma was taken from a rat that had been fed DAB for 114 days and normal food for 213 days. b For liver tissue differentiation, fetal was
