[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,

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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 phosphoribosyltransíerase; 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.

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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-

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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.

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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

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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.

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Enzymic Lesions of NAD Biosynthesis in Hepatomas

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MARCH 1967 583

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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

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