[CANCER RESEARCH 27 Part 1, 578-583, March...
Transcript of [CANCER RESEARCH 27 Part 1, 578-583, March...
[CANCER RESEARCH 27 Part 1, 578-583, March 1967]
Enzymic Lesions of Nicotinamide Adenine Dinucleotide Biosynthesisin Hepatomas and in Azo Dye Carcinogenesis1
MAKOTO SHIMOYAMA,2 KENJI YAMAGUCHI, AND ROBERT K. GHOLSON3
Oklahoma State University, Agricultural Experiment Station, Department of Biochemistry, Stillwater, Oklahoma 74075
SUMMARY
Several enzymes involved in nicotinamide adenine dinucleo-tide (NAD) biosynthesis were assayed in transplanted and azo-dye-induced primary hepatomas in order to determine the en-zymic lesion responsible for the low level of NAD present in thesetumors. The same enzymes were also investigated in precan-cerous livers of 3'-methyl-4-dimethylaminobenzene (3'-Me-DAB)fed rats and in rats receiving 3'Me-DAB and 4'-Me-DAB i.p.,as well as rats fed a-naphthyl isothiocyanate. It was concludedthat the low level of NAD in the tumors examined was probablydue to the deletion of enzymes in the tryptophan-to-NAD pathway and to decrease in activity of nicotinic acid mononucleotidepyrophosphorylase which catalyzes the rate-limiting step in thenicotinamide-to-NAD pathway. Changes in these enzymes inprecancerous liver did not satisfy Reid's criteria for key steps in
the carcinogenic process.
INTRODUCTION
The concentration of NAD4 and other pyridine nucleotides in
malignant tissues has been the subject of much recent investigation. The levels of the pyridine nucleotides in tumors are generallymuch lower than in the corresponding normal tissues (9, 19, 22,31, 39). A hypothesis linking this low level of NAD to the rapidcellular division characteristic of neoplastic tissue has been proposed by Morton (38, 39). On the other hand, a variety of carcino-static agents cause a drastic decrease in the concentration of NADin tumor cells (1, 11, 13-15, 45), and it has been proposed thatthis decrease in NAD level is responsible for the carcinostaticactivity of these compounds. In spite of the importance whichhas been attached to the concentration of NAD in tumors, verylittle has been done to establish the activity of the various enzymes responsible for NAD biosynthesis in normal and tumor
1 Supported in part by Grant No. GM-10066 from the NIH andby Grants No. GM-1659 and GB-4695 from the National ScienceFoundati on. A preliminary report of a portion of this work hasbeen published (46).
2 Present address, Department of Medical Chemistry, Osaka
Medical College, Osaka, Japan.3 Reprint requests should be addressed to this author.4 The abbreviations used are: NAD, nicotinamide adenine di-
nucleotide; 3'-Me-DAB, 3'-methyl-4-dimethylaminoazobenzene;4'-Me-DAB, 4'-methyl-4-dimethylaminoazobenzene; NMN, nico
tinamide mononucleotide; NAMN, nicotinic acid mononucleotide;3-OHAA, 3-hydroxyanthranilic acid.
Received July 5, 1966; accepted November 3, 1966.
tissue. Branster and Morton (2) found that NMN-adenyl trans-ferase activity of mouse mammary carcinoma is reduced to 20%of that of normal tissue, and projiosed that this enzymic lesionwas responsible for the low level of NAD in tumor tissues in general. Greenbaum et al. (12) also reported recently that NMN-adenyl transferase was reduced to approximately 50% of thenormal level in azo-dye-induced primary hepatomas. The nicotinamide deamidase activity of a number of tumors has been reported recently (32), but no direct comparison was made withthe corresponding normal tissues. Several enzymes related toNAD biosynthesis have been discovered since the investigationsof Branster and Morton (2). We have compared the activity ofa number of these enzymes in normal rat liver, in precancerousliver of azo-dye-fed rats and in primary and transplanted hepatomas in order to determine whether NMN-adenyl transferaseis actually the rate-limiting step in the deficient NAD biosynthesis of tumor tissue.
MATERIALS AND METHODS
Animals. Male rats were obtained from Holtzman Rat Co.'Madison, Wisconsin. These animals weighed 100-150 gm at thebeginning of each experimental series.
Diets. The basal or control diet was the vitamin B complextest diet obtained from Nutritional Biochemicals, Inc., supplemented with vitamins to give a riboflavin-deficient diet (0.5 mg/kg of riboflavin) as described by Medes et al. (34). Pair-fed controls were used with the azo-dye- and a-naphthyl isothiocyanate-fed animals. 3'-Me-DAB and 4'-Me-DAB were synthesized fromappropriate precursors by the method of Giese et al. (S), a-Naphthyl isothiocyanate was purchased from K and K Laboratories, Inc. The azo dyes were added to the basal diet to give aconcentration of 0.06% and a-naphthyl isothiocyanate was fedat a level of 0.1%. Food intake of the control animals was restricted to equal that of the azo dye or a-napthyl isothiocyanate-fed animals. In experiments in which azo dyes were injected, 3ml of corn oil in which 40 mg of azo dye were dissolved by heatingto 60-65°Cwere injected i.p. Control animals were injected with
3 ml of the warm corn oil.Transplantable Tumors. The Novikoff hepatoma and
another transplantable hepatoma induced at the Samuel RobertsNoble Foundation by feeding 3'-Me-DAB (hereafter referred to
as DAB hepatoma) were obtained from Dr. D. E. Kizer of theSamuel Roberts Noble Foundation, Ardmore, Oklahoma. Thesetumors were maintained by intramuscular transplantation.
Enzyme Assays. Rats were killed by cervical dislocation andthe livers or tumors removed, perfused with ice-cold 0.9r'~/osaline,
578 CANCER RESEARCH VOL. 27
on March 6, 2020. © 1967 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from
Enzymic Lesions of XAD Biosynthesis in Hepatomas
XAD
TryptophanI"
Formyl kynurenine
IKymirenine
I63-Hydroxvkvnnrenine
Ie3-OHAA Desamido-XAD
I" , -Î2-Aniino-3-carboxvmucomc semialdehvde—>Giiinolimc acid—->XAMX
1- »T2-Aminomuconic aemialdehyde —>Picoliuic acid Xicotinic acid
¿' ;î2-Aminomuoonio acid —>f!lutaryl coenzyme A—>('Os Xicotinamidc
SCHEMEI. Pathways of NAD biosynthesis. Enzymes: a, trypto-phan pyrrolase (EC 1.13.1.12); 6, kynurenine hydroxylase (EC1.14.1.2); c, kynureninase (EC 3.7.1.3); d, 3-hydroxyanthranilicacid oxidase (EC 1.13.1.6); e, picolinic carboxylase; /, 2-aminomu-conic semialdehyde dehydrogenase; g, quinolinate phosphoribosyltransÃerase; h, nicotinic acid mononucleo tide pyrophosphorylase(EC 2.4.2.11); i, NMN-adenyltransferase (EC 2.7.7.1); j, nicotin-amide deamidase.
TABLE 1Levels of 8-Hydroxyanthranilic Acid Oxidase and a-Aminomuconic
Semialdehyde Dehydrogenase in Normal Rat Liver andHepatomas"
EnzymesourceNormal
liverPrimaryhepatoma6timóles
product/min/mg protein(X101)at27°C3
-Hydroxyan thranilicacidoxidase152.5
±12.45.5±1.7Liver
tissue adjacent to 102.5 ±6.5hepatomaNovikoff
hepatoma'Livers
of Novikoff hepatoma rats<0.1145.0
±2.5a-Aminomuconic
semialdehydedehydrogenase9.0
±1.31.0±0.46.8±0.7<0.17.8
±0.3
" Experimental conditions are described in the text. Values are
the means from enzyme assays on tissues of 4 rats ±S.E.c Induced by feeding 3'-methyl-4-dimethylaminoazobenzene.e Obtained from Dr. D. E. Kizer, Samuel Roberts Noble Foun
dation, Ardmore, Oklahoma, and maintained by i.m. transplantation.
and homogenized in 4 volumes of water at 0°Cin a Potter ho-mogenizer. NMN-adenyl transferase activity was assayed onaliquota of the homogenate before centrifugation by the methodof Branster and Morton (2). The homogenate was then centri-fuged at 20,000 X g for 30 min and aliquota of the supernatantwere assayed for 3-OHAA oxidase (5), a-aminomuconic semi-aldehyde dehydrogenase (6), quinolinate phosphoribosyl transferase, and (7) NAMN pyrophosphorylase (18) as described inthe literature. Picolinic carboxylase was assayed by the spectro-photometric method of Mehler (35). Protein concentration wasdetermined by the method of Lowry et al. (30).
RESULTS
Enzyme Levels in Hepatomas
In most animals, including rats, two pathways are availablefor the biosynthesis of NAD, the tryptophan-to-XAD pathway,and the more direct route from dietary niacin or niacinamide (21).Previous work (26, 41) demonstrated that the early enzymes ofthe tryptophan pathway (see Scheme I) tryptophan pyrrolase(a), kynurenine hydroxylase (6), and kynureninase (c) were absent from azo-dye-induced and Novikoff hepatomas. However, nostudies have been reported on the later enzymes of thetryptophan-NAD pathway, and it is possible that intermediatesformed in normal tissue could be transferred to tumor cells andthere converted to NAD. Therefore, some of the terminal enzymes of tryptophan metabolism were investigated in severalhepatomas with the results shown in Table 1.
3-OHAA oxidase (d), an enzyme required for the conversionof tryptophan to NAD via quinolinate, is greatly reduced in activity in primary hepatomas and completely absent in the Novikoff hepatoma. Picolinic carboxylase (e), the enzyme which initiates the oxidative (or glutarate) pathway of tryptophancatabolism (6, 16) is of such low activity in normal rat liver as tobe almost undetectable by the spectrophotometric assay (35) em-
TABLE 2Levels of Three Enzymes of XAD Biosynthesis in Normal Rat
Liver and Transplanted Hepatomas^
/imoles product formed/min/mg protein (Xl</) at 37°C
EnzymesourceNormal
liver[7]Novikoffhepatoma[6]"DAB"*
hepatoma[6]Liver
ofNovikofftumorrats[6]Livers
of DAB tumor rats [6]Qutnolinate
phosphoribosyl-transferase89.3
±5.20091.7
±8.866.7
±5.8Nicotinic
acidmononucleotidepyrophosphorylase56.0
±7.85.8±0.53.7
±0.352.8
±7.250.0
±3.3NMN-adenyl
transferaseft553.3
±30.0358.3±35.Oc358.3
±35.0«508.3
±46.7«516.7
±6.7C
°Experimental conditions are described in the text. Numbers
in brackets refer to number of animals assayed in each group.Values are average ±S.E.
*NMN, nicotinamide mononucleotide; DAB, dimethylamino-azobenzene.
e Two animals.
ployed in these studies. However, it has been recently reported(17, 36) that the "block" in the conversion of tryptophan to
niacin observed in diabetes is actually due to a sharp increase inpicolinic carboxylase activity which depletes 2-amino-3-car-boxymuconic semialdehyde, the common intermediate of theglutarate and NAD pathways of tryptophan metabolism. Nosuch increase in picolinic carboxylase occurs in hepatomas,ruling out this mechanism for decreased NAD formation. Theactivity of a-aminomuconic semialdehyde dehydrogenase (/),another enzyme of the glutarate pathway, is also greatly reducedin primary hepatoma and lost in the Novikoff tumor.
MARCH 1967 579
on March 6, 2020. © 1967 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from
Makoto Shimoyama, Kenji Yamaguchi, and Robert K. Gholson
TABLE 3Activity of Enzymes Related to NAD Biosynthesis in 3'-Me-DAB"-induced Hepatomas0
EnzymeNormal
liverPrimary hepatomaLiver adjacent to
hepatomaMmoles/min/mg
protein (X103) at27°C3-OHAA
oxidase145.0
±7.55.9
± 1.385.0 ± 4.5a-AMS
dehydrogenase8.7
± 2.20.8 ±0.38.5 ± 0.6iimoles/min/mg
protein (X10e) at37°CQA
phosphoribosyltransferase68.3
±3.365.0 ±5.081.3 ±5.0NAMX
pyro-phosphorylase35.0
± 3.321.7 ±5.036.7 ± 10.0NMN-adenyl
transferase541.7
±16.7375.0 ±41.7500.0 ±41.7
" 3'-Me-DAB, 3'-methyl-4-dimethyIaminoazobenzene; 3-OHAA, 3-hydroxyanthranilic acid; AMS>a-aminomuconic semialdehyde; QA, quinolinate; NAMN, nicotinic acid mononucleotide; NMN, nico-tinamide mononucleotide.
6Experimental conditions are described in the text. The average weight of rats in all groups wasapproximately 450 grams. Values are the means for enzyme assays on tissues of 5 rats ±S.E.
A comparison of the levels in normal liver and transplantedhepatoma of three enzymes directly involved in NAD biosynthesis, quinolinate phosphoribosyl transferase (Keaction g),NAMN pyrophosphorylase (Reaction /;), and NMN-adenyltransferase (Reaction i) is shown in Table 2. The specific activity of NMX-adenyl transferase in the transplanted hepatomasis about two-thirds that of normal liver. However, since thetumor tissue contains less protein than normal liver, the totalactivity per gm wet weight in these transplantable hepatomas isabout one-third that of normal liver; this agrees with previousfindings (2, 4). A n:ore striking decrease is observed in the activities of quinolinate phosphoribosyl transferase and NAMNpyrophosphorylase, the enzymes which synthesize NAMN. Theformer appears to be completely absent from the transplantedhepatomas studied while the latter is reduced to 10% or less ofits activity in normal liver. It appears, therefore, much morelikely that the rate-limiting step in NAD biosynthesis in the DABand Novikoff hepatomas is the formation of NAMN rather thanthe condensation of this compound with adenosine triphosphatecatalyzed by NMN-adenyl transferase.
The profile of NAD-related enzymes in primary hepatomasinduced by feeding 3'-Me-DAB is shown in Table 3. This pro
file differs in some respects from that of Novikoff and DABhepatomas. 3-OHAA oxidase and a-aminomuconic semialdehydedehydrogenase are reduced to 10% or less of their activity innormal tissue and NMN-adenyl transferase specific activity isabout two-thirds of normal tissues, as was observed in the transplantable hepatomas. However, NAMN pyrophosphorylase activity is reduced to 60% of normal, and quinolinate phosphori-bosyltransferase activity is unchanged as contrasted to the drasticreduction of these enzyme activities seen in transplantablehepatomas. Picolinic carboxylase activity could not be detectedin primary hepatomas.
Activity of NAD-synthesizing Enzymes in PrecancerousLiver
The changes in activity of various hepatic enzymes during azodye carcinogenesis have been studied by a number of investigators in the hope of identifying critical enzymic lesions in the development of neoplasia (3, 23-25, 28, 43, 48). The data presented
in Table 4 show the changes in the activity of several enzymes related to NAD biosynthesis during 12 weeks of feeding with 3'-Me-
DAB and the basal diet. Pair feeding with the control diet for8-12 weeks caused significant reductions in the activity of a-
aminomuconic semialdehyde dehydrogenase and NAMN pyrophosphorylase, suggesting that the level of these enzymes is depressed by restricted food intake. During the early weeks offeeding the 3'-Me-DAB diet, 3-OHAA oxidase and NAMN pyro
phosphorylase activities were greatly reduced from control levelsas shown in Table 4. The activities of the other enzymes were notappreciably affected during 12 weeks. It is known that feeding of3'-Me-DAB results in extensive and progressive bile duct pro
liferation, e.g., Ref. 48, and it was possible that reduction ofcertain enzyme activities in azo-dye-fed liver represented a relative increase in the population in bile duct cells, which cells maynot contain the enzyme under consideration. It has been demonstrated that feeding a-naphthyl isothiocyanate results in massivebile duct hyperplasia (29,33,47). However, this compound is not ahepatocarcinogen (33). Therefore, experiments were carried outto determine if feeding a-naphthyl isothiocyanate had any effecton the activity of 3-OHAA oxidase and NAMN pyrophosphorylase. The results of these experiments are shown in Charts1 and 2. The activity of these two enzymes was decreased byfeeding a-naphthyl isothiocyanate to about the same extent asproduced by feeding 3'-Me-DAB. The level of NAMN pyrophos
phorylase in the a-naphthyl isothiocyanate controls did not decline as in the 3'-Me-DAB controls. This is probably due to asignificantly larger food intake in the a-naphthyl isothiocyanatecontrol group.
Recently, it has been discovered that a single intraabdominalinjection of 3'-Me-DAB will produce changes in the liver in a few
days similar to those produced by weeks of feeding the dye (10,20, 27, 40). These alterations were not observed when the veryweakly carcinogenic 4'-Me-DAB was injected under the sameconditions (27, 40). The effects of i.p. injection of 3'-Me-DABand 4'-Me-DAB on the activity of some of the hepatic enzymes
concerned with NAD biosynthesis are shown in Table 5. Theactivities of a-aminomuconic semialdehyde dehydrogenase,quinolinate phosphoribosyl transferase, and NAMN pyrophosphorylase were greatly reduced in 3'-Me-DAB-injected rats ascompared to 4'-Me-DAB-injected animals. The oil injection it-
580 CANCER RESEARCH VOL. 27
on March 6, 2020. © 1967 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from
Enzymic Lesions of NAD Biosynthesis in Hepatomas
TABLE 4The Activities of Enzymes Related to NAD Biosynthesis in Livers of Rats Fed S'-Me-DAB "•b
ro•S3
"9|l024681012ExperimentalconditionControl3'-Me-DABControl3'-Me-DABControl3'-Me-DABControl3'-Me-DABControl3'-Me-DABControl3'-Me-I)ABNo.of
rats5454544444445Bodyweightaverage
(gm)127138125136132180130200156200156169148jimoles
product/min/mg protein (X103) at27°C3-OHAA
oxidase197.0
±3.6195.0
±6.1173.0±5.0237.5±7.2125.0±18.4185.0±7.9114.7±10.6202.5±2.2102.5±15.2215.0±9.4122.5±6.5178.3±2.7115.0± 8.9a-AMS
dehydrogenase10.30
±0.669.50±0.358.50±0.4910.00±0.835.75±1.6310.00±0.756.00±0.437.00±0.186.00±0.836.17±0.276.75±0.224.09±0.294.00±0.25Amóles
product/ m¡n/protei n (XNV) at37°CQA
phosphoribosyltransferase128.3
±3.0104.2±4.5108.3±6.791.7±2.291.7±13.2104.2±7.095.8±3.762.5±4.279.2±7.269.5±4.579.2±5.562.5±3.755.0± 6.0NAMN
pyrophos-phorylase55.0
±3.029.2±3.715.0±3.533.3±4.312.5±3.733.3±4.320.8±4.312.5±3.78.3±3.713.8±4.320.8±3.713.8±4.318.3±3.7NMN-adenyl
transferase750.0
±66.7583.3±58.3580.0±55.0750.0±58.3666.7±58.3666.7±41.7750.0±58.3805.0
±41.7570.0±40.0756.7±25.0616.7±30.0
" The experimental details are described in the text. Values are averages of assay for livers of number of rats indicated ±S.E.6Abbreviations: see Footnote a, Table 3.
ÃœJ
C£CL
CO UJ
Xo
2005
100-
orO
COÃœJ
o
ÃŽ8
WEEKSCHART1. The hepatic 3-hydroxyanthranilic acid (3-OHAA)
oxidase activity of rats fed a-naphthyl isothiocyanate for18 weeks. a-Naphthyl isothiocyanate was fed at 0.1%. Each pointrepresents averages of assays of five individual rat livers. Verticallines on either side of each point designate standard error.
self appeared to lower significantly the activity of 3-OHAA oxidase, quinolinate phosphoribosyl transferase, and NAMN pyro-phosphorylase, while NMN-adenyl transferase activity was notgreatly affected by oil or either azo dye.
DISCUSSION
The main purposes of this investigation were to pini>oint theenzymic lesion responsible for the comparatively low level ofNAD which is found in a variety of tumors, and to determine ifany of the enzymic changes observed might be a key change inthe carcinogenic process.
The data presented in Tables 1-3 do not support the earliersuggestion of Branster and Morton (2) that decreased activity of
8 12 16
WEEKS
CHABT2. The hepatic nicotinic acid mononucleotide (NAMN)pyrophosphorylase activity of rats fed a-naphthyl isothiocyanatefor 18 weeks. a-Naphthyl isothiocyanate was fed at 0.1%. Eachpoint represents averages of assays on five individual rat livers.Vertical lines on either side of each point designate standard error.
NMN-adenyl transferase is the reason for the low NAD concentration of tumors. These data rather support the conclusion that,in the hepatomas examined, a decreased rate of NAD synthesisis due to deletion of enzymes on the tryptophan to NAD pathway and reduction of the activity of NAMN pyrophosphorylase,an enzyme of the nicotinamide-to-NAD pathway. Reduction ofenzyme activity will only decrease product formation if that enzyme is already the rate-limiting step in a biosynthetic sequence(or becomes the rate-limiting step after decrease in activity). Ithas been proposed on the basis of the low activity of this enzymein in vitro assays that nicotinamide deamidase (j) (Scheme I) isthe rate-limiting step in NAD biosynthesis from nicotinamide inliver (42) and Ehrlich ascites tumor cells (32). However, other
MARCH 19G7 581
on March 6, 2020. © 1967 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from
Makolo Shimoyama, Kenji Yamaguchi, and Robert K. Gholson
TABLE 5Response of Hepatic Enzymes Related to NAD Biosynthesis to Inlraperitoneal Injection of S'-Me-DAB
and t'-Me-DAB'- »
Daysafterinjection0222444ExperimentalconditionsControl3'-Me-DAB4'-Me-DABControl3'-Me-DAB4'-Me-DAB/imoles/rnin/mg
protein (X10>)at27°C3-OHAA
oxidase197.0
±3.4127.0
±9.9115.0±8.0125.0±2.8115.0
±5.795.0±6.3115.0±6.3a-AMS
dehydro-genase10.36
±0.6611.90
±0.456.10±0.2210.10±0.6111.30
±0.662.70±0.189.
.50 ±0.72^moles/min/mg
protein (X10S)at37°CQA
Phospho-ribosyltransferase128.3
±3.061.7
±3.065.0±3.761.7±3.051.7
±3.728.3±3.051.7±3.7NAMN
pyro-phosphorylase55.0
±3.041.7
±4.725.0±4.735.0±7.730.5
±6.013.8±4.535.0±6.8NMN-adenyl
transferase750.0
±66.7616.7
±85.0750.0±38.3676.7±55.0716.7
±30.0883.3±55.0816.7±76.7
" Experimental details described in the text. Values are averages of assays for livers of 5 rats ±S.E.6Abbreviation: see Footnote a, Table 3.
lines of evidence indicate that, at least in normal liver, XAMNpyrophosphorylase is the rate-limiting enzyme in this pathway.
It has been reported that free nicotinic acid accumulates in theliver of nicotinamide-injected rats (44); this would not occur ifnicotinamide deamidase were the rate-limiting step. It has also
been reported that the level of hepatic NAD increases muchmore rapidly after injection of NAMN than of either nicotinicacid or nicotinamide (37), again suggesting that NAMN pyrophosphorylase is the rate-limiting enzyme in NAD biosynthesisfrom nicotinamide. It therefore ap)x>ars plausible that the ob
served decrease in the activity of this enzyme in hepatomascoupled with the deletion of the tryptophan-NAD pathway
could account for the observed low level of NAD in these tumors.Reid (43) has suggested three criteria to determine which bio
chemical effects of hepatocarcinogens are significant steps toward neoplasia. The first two of these are: (a) key changes willlie at steps which are rate limiting in normal liver; (6) any effectproduced by diverse carcinogens but not by noncarcinogenicanalogs is likely to be a key change. As has been discussed above,NAMN pyrophosphorylase meets the first criterion since itcatalyzes what is probably the rate-limiting step in the formation
of NAD from nicotinamide. The activity of this enzyme is alsoreduced upon feeding of 3'-Me-DAB (Table 4) and upon intra-peritoneal injection of 3'-Me-DAB (Table 5) but not upon injection of the weakly carcinogenic 4'-Me-DAB (Table 5). How
ever, NAMN pyrophosphorylase activity is also greatly decreasedby feeding the noncarcinogenic a-naphthyl isothiocyanate
(Chart 2), suggesting that this decrease may be due to bile ducthyperplasia rather than being a step in hepatocarcinogenesis.
It may be concluded from the results of this study that thedeletion of the tryptophan-NAD pathway together with a de
creased activity of NAMN pyrophosphorylase is a possible explanation for the reduced level of NAD observed in hepatomas.However, the decrease in NAMN pyrophosphorylase activitydoes not appear to be a significant change in carcinogenesis asdefined by Reid's criteria (43).
ACKNOWLEDGMENTS
We are most grateful to Dr. Donald E. Kizer for providing theNovikoff and DAB hepatomas and for his frequent advice andencouragement during the course of these investigations. Thecontributions of Mr. S. J. Lan to the early phases of this work andthe competent technical assistance of Miss K. Usuki are also gratefully acknowledged.
REFERENCES
1. Barclay, R. K., and Phillipps, M. A. Effects of 6-Diazo-5-oxo-L-norleucine and Other Tumor Inhibitors on the Biosynthesisof Nicotinamide Adenine Dinucleotide in Mice. Cancer Res.,26: 282-286, 1966.
2. Branster, M. J., and Morton, R. K. Comparative Rates ofSynthesis of Diphosphopyridine Nucleotide by Normal andTumor Tissue from Mouse Mammary Gland: Studies withIsolated Nuclei. Biochem. J., 65: 640-646, 1956.
3. Cantero, A. Metabolic Changes in Precancerous Tissue. Can.Cancer Conf., 1: 309-318, 1955.
4. Clark, J. B., Greenbaum, A. L., McLean, P., and Reid, E.Concentration and Rates of Synthesis of Nicotinamide-ade-nine-dinucleotide Phosphate in Precancerous Livers andHepatomas Induced by Azo-dye Feeding. Nature, SOI: 1131-1132, 1904.
5. Decker, R. H., Kang, H. II., Leach, F. R., and Henderson,L. M. Purification and Properties of 3-Hydroxyanthranilic AcidOxidase. J. Biol. Chem., 236: 3076-3082, 1961.
6. Gholson, R. K., Nishizuka, Y., Ichiyama, A., Kawai, H.,Nakamura, S., and Hayaishi, O. New Intermediates in theCatabolism of Tryptophan in Mammalian Liver. J. Biol.Chem., 237: PC2043-PC2045, 1962.
7. Gholson, R. K., Ueda, I., Ogasawara, N., and Henderson,L. M. The Enzymatic Conversion of Quinolinate to NicotinicAcid Mononucleotide in Mammalian Liver. J. Biol. Chem.,239: 1208-1214,1964.
8. Giese, J. E., Miller, J. A., and Baumann, C. A. The Carcino-genicity of TO-Methyl-jo-dimethylaminoazobenzene and p-Monomethylaminoazobenzene. Cancer Res., 5: 337-340, 1945.
9. Clock, G. E., and McLean, P. Levels of Oxidized and ReducedDiphosphopyridine Nucleotide and Triphosphopyridine Nucleotide in Tumors. Biochem. J., 65: 413-416, 1957.
10. Green, H. N., and Chose, T. Localization of Liver Specific
5X2 CANCER RESEARCH VOL. 27
on March 6, 2020. © 1967 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from
Enzymic Lesions of NAD Biosynthesis in Hepatomas
Antibody in Normal and 3'-DAB-treated Rats. Nature, a/0:308-309, 1964.
11. Green, S., and Bodansky, O. Effect of MethylbisGS-chloro- 28.ethyl)amine (Nitrogen Mustard) on the DiphosphopyridineNucleotidase Activity of Ehrlich Ascites Cells and the Roleof This Effect ia Glycolysis. J. Biol. Chem., 237: 1752-1757, 29.
1962.12. Greenbaum, A. L., Clark, J. B., and McLean, P. The Rates of
Synthesis and Tissue Concentrations of Pyridine Nucleotidesas Metabolic Regulators. Proc. Sixth Intern. Biochem. Cong., 30.New York Abstracts, IX: 719, 1964.
13. Hilz, H., Hlavica, P., and Betram, B. Die entscheidendeBedeutung der DPNase-Aktivierung fürden DPN-Abfall in 31.bestrahlter Ascitestumorzellen. Biochem. Z., 33S: 282-299,
1963.14. Holzer, H., Glongner, P., and Sedlmayr, (!. Zum Mechanismus
der Glykolysehemmung durch carcinostatisch wirkende 32.Athyleniminverbindungen. Biochem. Z., SSO:59-72, 1958.
15. Holzer, H., and Kroger, H. Zum carcinostatischen wirkungs-mechanismus von Athylenimin-Verbindungen. Biochem. Z., 33.330: 579-590, 1958.
16. Ichiyama, A.,Nakamura, S.,Kawai,H., Honjo,T.,Xishizuka,Y., Hayaishi, O., and Senoh, S. Studies on the Metabolism ofthe Benzene Ring of Tryptophan in Mammalian Tissues. II. 34.Enzymic Formation of a-Aminomuconic Acid from 3-Hydroxy-anthranilic Acid. J. Biol. Chem., 240: 740-749, 1965.
17. Ikeda, M., Tsuji, H., Nakamura, S., Ichiyama, A, Nishizuka,Y., and Hayaishi, O. Studies on the Biosynthesis of Nicotin- 35.amide Adenine Dinucleo tide II. A Role of Picolinic Carboxylasein the Biosynthesis of Nictoinamide Adenine Dinucleotidefrom Tryptophan in Mammals. J. Biol. Chem., 240:1395-1401, 36.
1965.18. Imsande, J. Pathway of Diphosphopyridine Nucleotide Bio
synthesis in Escherichia coli. J. Biol. Chem., 236: 1494-1497, 37.
1961.19. Jedeikin, L. A., and Weinhouse, S. Metabolism of Neoplastic
Tissue. VI. Assay of Oxidized and Reduced Diphosphopyridine 38.Nucleotide in Normal and Neoplastic Tissues. J. Biol. Chem.,SIS: 271-280, 1955.
20. Jinnohara, T. Histological and Histochemical Studies of the 39.Early Changes of Rat Liver Cells during 3'-Methyl-4-dimeth-
ylaminoazobenzene Feeding and its Intraperitoneal Injection. 40.Sapporo Igaku Zasshi, 23: 252-269, 1963 (original not seen);
Carcinogenesis Abstr., 2: 886, 1964. 41.21. Kaplan, N. O. Metabolic Pathways Involving Niacin and its
Derivatives. Metabolic Pathways, 2: 627-672, 1961.22. Kensler, C. J., Sugiura, K., and Rhoads, C. P. Coenzyme I 42.
and Riboflavin Content of Livers of Rats Fed Butter Yellow.Science, 91: 623, 1940.
23. Kizer, D. E. Relationships between Hepatocarcinogenesis and 43.the Precancerous Loss of 5-Hydroxytryptophan Decarbox-ylase Activity. Cancer Res., 82: 196-201, 1962. 44.
24. Kizer, D. E. Precancerous Changes in the Activity of EnzymesAssociated with Tryptophan Metabolism and their Relevancyto Hepatocarcinogenesis. Ann. N. Y. Acad. Sci., IOS: 1127- 45.1135, 1963.
25. Kizer, D. E., and Chan, S. K. The Effect of Hepatocarcinogenesis upon 5-Hydroxytryptophan Decarboxylase and Sero- 46.tonin Deaminase. Cancer Res., HI: 489-495, 1961.
26. Kizer, D. E., and Howell, B. A. The Deletion of KynurenineHydroxylase Activity during Hepatocarcinogenesis. J. Nati. 47.Cancer Inst., 30: 675-686, 1963.
27. Kizer, D. E., Howell, B. A., Shirley, B. C., Clouse, J. A., andCox, B. Effect of Intraabdominal Injections of 3'-Methyl-4- 48.
dimethylaminoazobenzene on Adenylic Acid Deaminase Activ
ity and Nuclear Ribonucleic Acid Metabolism of Rat Liver.Cancer Res., 26: 822-828, 1966.
Kizer, D. E., Lovig, A. C., Howell, B. A., and Cox, G. Changesin the Adenylic Acid Deaminase Activity of Rat Liver duringCarcinogenesis. Cancer Res., 24: 1050-1055, 1964.Lopez, M., and Mazzanti, K. Experimental Investigations onAlpha-Naphthyl-isothiocyanate as a Hyperplastic Agent ofthe Biliary Ducts in the Rat. J. PathoÃ. Bacteriol., 69: 243-250, 1955.Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall,R. J. Protein Measurement with the Folin Phenol Reagent.J. Biol. Chem., 193: 265-275, 1951.Mandel, P., Wintzererith, M., Klein-Pete, N., and Mandel, L.Comparative Investigation of the Free Nucleotides of anAscitic Hepatoma and of Normal or Regenerating Liver. Nature, 198: 1000-1001, 1963.Marki, F., and Greengard, P. Nicotinamide Deamidase inEhrlich Ascites Tumor Cells. Biochim. Biophys. Acta, 113:587-594, 1966.
McLean, M. R., and Rees, K. R. Hyperplasia of Bile DuctsInduced by 1-Napthyl Isothiocyanate. Experimental BiliaryCirrhosis Free From Biliary Obstruction. J. Pathol. Bacteriol.,76: 175-188, 1958.
Medes, G., Friedman, B., and Weinhouse, S. Fatty Acid Metabolism. VIII. Acetate Metabolism in Vitro during Hepatocarcinogenesis by p-Dimethylaminoazobenezene. Cancer Res.,16: 57-62, 1956.
Mehler, A. H. Formation of Picolinic and Quinolinic AcidsFollowing Enzymatic Oxidation of 3-HydroxyanthranilicAcid. J. Biol. Chem., 218: 241-254, 1956.Mehler, A. H., Yano, K., and May, E. L. Nicotinic Acid Biosynthesis: Control by an Enzyme that Competes with a Spontaneous Reaction. Science, 145: 817-819, 1964.
Minard, F. N., and Hahn, C. H. Efficacy in Vivo of Precursorsof Nicotinamide Adenine Dinucleotide. J. Biol. Chem., 238:2474-2476, 1963.Morton, R. K. Enzymic Synthesis of Coenzyme I in Relationto Chemical Control of Cell Growth. Nature, 181: 540-542,
1958.Morton, R. K. New Concepts of the Biochemistry of the CellNucleus. Australian J. Sci., 24: 260-278, 1961.Neish, W. J. P., and Rylett, A. Azo Dyes and Rat Liver Gluta-thione. Biochem. Pharmacol., 12: 893-903, 1963.
Petasnick, J. P., Brown, R. R., and Price, J. M. The Effect ofFeeding 3'-Methyl-4-dimethylaminoazobenzene on Tryptophan Metabolism of Rats. Cancer Res., 21: 1400-1405, 1961.Petrack, B., Greengard, P., Cranston, A., and Kalinsky, H. J.Nicotinamide Deamidase in Rat Liver and the Biosynthesisof NAD. Biochem. Biophys. Res. Commun., 13: 472-477, 1963.Reid, E. Significant Biochemical Effects of Hepatocarcinogenesis in the Rat: A Review. Cancer Res., 22: 398-430, 1962.Ricci, C., and Pallini, V. Occurrence of Free Nicotinic Acid inthe Liver of Nicotinamide-injected Rats. Biochem. Biophys.Res. Commun., 17: 34-38, 1964.Roitt, I. M. The Inhibition of Carbohydrate Metabolism inAscites-Tumour Cells by Ethyleneimines. Biochem. J., 63:300-307, 1956.Shimoyama, M., Kori, J., Usuki, K., Lan, S. J., and Gholson,R. K. Enzymic Lesions of NAD Biosynthesis in Hepatomas.Biochim. Biophys. Acta, 97: 402-404, 1965.Steiner, J. W., and Carruthers, J. S. Electron Microscopy ofHyperplastic Ductular Cells in a-Naphthyl Isothiocyanate-induced Cirrhosis. Lab. Invest., 12: 471-498, 1963.Weber, G. Behavior of Liver Enzymes in Hepatocarcinogenesis. Advan. Cancer Res., 6: 403-494, 1961.
MARCH 1967 583
on March 6, 2020. © 1967 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from
1967;27:578-583. Cancer Res Makoto Shimoyama, Kenji Yamaguchi and Robert K. Gholson Biosynthesis in Hepatomas and in Azo Dye CarcinogenesisEnzymic Lesions of Nicotinamide Adenine Dinucleotide
Updated version
http://cancerres.aacrjournals.org/content/27/3/578
Access the most recent version of this article at:
E-mail alerts related to this article or journal.Sign up to receive free email-alerts
Subscriptions
Reprints and
To order reprints of this article or to subscribe to the journal, contact the AACR Publications
Permissions
Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)
.http://cancerres.aacrjournals.org/content/27/3/578To request permission to re-use all or part of this article, use this link
on March 6, 2020. © 1967 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from