(CANCER RESEARCH 34, 2489-2494, October 1974 ]

Early Elevation of oîi-Fetoproteinin Af-2-FluorenylacetamideHepatocarcinogenesis1

Frederick F. Becker and Stewart Sell

Department of Pathology, New York University School of Medicine, New York, New York 10016 [F. F. B.], and Department of Pathology,The University of California School of Medicine, San Diego, California 92037 [S. S.]

SUMMARY

Utilizing a radioimmunoassay capable of detecting ngquantities of circulating rat o^-fetoprotein, we examined theinduction of this protein by TV-2-fluorenylacetamide. Extremely small quantities of ,/V-2-fluroenylacetamide, approximately 1% of a carcinogenic dose, caused a rapid andsignificant elevation of «i-fetoprotein that persisted for aconsiderable period after removal of the carcinogen from thediet. Although the most rapid and often highest elevationswere achieved in male rats, comparable elevations wereeventually demonstrated in female rats of strains that hadselective defects in the metabolic activation of the carcinogen.The elevation in all groups preceded any detectable tissuealteration and was apparently unrelated to cell injury or celldivision.

We suggest that the increase in circulating c^-fetoproteinseen after minimal jV-2-fluorenylacetamide exposure is theresult of a highly selective derepression of protein synthesisthat occurs following the formation of a complex between themetabolite(s) of the carcinogen and specific chromatin loci.

INTRODUCTION

a¡F,2 the dominant plasma protein elaborated by the fetal

liver, is also synthesized by a number of human and animalhepatomas (1, 3,5,18,21,23). Previous studies have reportedthe appearance of elevated levels of atF during exposure ofrats to several hepatocarcinogens and have attempted tocorrelate this phenomenon with the chemical structure of theagents administered, with their carcinogenicity, with theaccumulated dose, and with histológica! tissue alterations(13-15, 17, 22, 25, 26). A major problem in the interpretation of these results is the relative insensitivity of the test(s)used to determine c^F levels and, in some instances, thespecificity of the antibody preparations. This insensitivityraises the possibility that elevations of c^F that might haveoccurred during the early phases of exposure to carcinogenwere undetected. A failure to detect these lower levels wouldalter the significance of correlations between circulating o^Fand tissue alterations.

'Supported by NIH Research Grant CA-12141 and ContractVO-l-CP-33403.

"The abbreviations used are: a, F, a, fetoprotein; 2-I-'AA, /V-2-fluo-

renylacetamide; SGPT, serum glutamic pyruvate transaminase.Received April 15, 1974; accepted June 11, 1974.

The development of a radioimmune inhibition assay ofextreme sensitivity and specificity (18, 19) has enabled us toexamine the level of a{ F in the period between the onset ofadministration of 2-FAA and the development of histologicaltissue alterations. The serum concentration of at F in normaladult rats is 30 ng/ml, and slight elevations above this level arereadily detectable (19). The serum a, F concentration of SDmale rats, which are fully susceptible to hepatocarcinogenesiswith 2-FAA, and the serum c*iF of female rats of 2 strains thathave selective defects in the metabolic activation of 2-FAAwere studied for induction of a} F production during 2-FAAadministration (7, 11, 12).

MATERIALS AND METHODS

Rats. SD rats were obtaind from 2 sources: CFE, fromCarworth Farms, New City, N. Y.; and TAC:NIH (SD) fBR,from Taconic Farms, Germantown, N. Y. No difference wasdetected in either of these strains in terms of a¡F induction orhistological response to 2-FAA. F/344 female rats wereobtained from Charles River Breeding Laboratory, Wilmington, Mass. In all of these experiments, the rats were received at100 to 110 g body weight and were placed on diets containingcarcinogens when attaining a weight of 125 g. Strain, weight,and sex-paired controls were utilized in all experiments todetermine the basal level of circulating CK|F. Previous studieshave demonstrated the importance of this measurement, asthere is a rapidly declining level in young rats while largeadults demonstrate a relatively stable basal level (20).

2-FAA was obtained from Eastman Organic Chemicals,Rochester, N. Y. This was mixed into a standard laboratorymeal as previously described (24). Rats were acclimatized to a "meal" diet by at least 1 week of feeding from

the time of receipt to onset of the carcinogen regimen.Intermittent carcinogen feeding schedules, as previouslyreported (24), were 3 weeks on carcinogen diet with 1 week ofnormal meal intervening. Continuous diet was administered ina standard manner. To prepare the diets containing extremelylow levels of 2-FAA, dilutions of a more concentratedcarcinogen diet were prepared in a mechanical mixing device.Details of the individual dietary programs will be included in"Results."

Blood was obtained by 2 methods. For repeated bleedings, aretroorbital pipetting was performed which enabled us toobtain between 0.5 and 1 ml of blood per rat. Blood obtainedat sacrifice was obtained via the inferior vena cava. All blood

OCTOBER 1974 2489

on May 14, 2018. © 1974 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

F. F. Becker and S. Sell

was collected in sodium oxalate and spun in the cold to collectthe supernatant plasma. Comparison of oti F concentrations inplasma obtained by the 2 methods revealed no difference.

atf. The plasma atF concentrations were measured usingthe radioimmunoassays described previously (18, 19). Allplasma samples were given code numbers by F. F. Becker andsent to S. Sell for assay. at F concentrations were recorded forthe coded samples and returned to F. F. Becker for analysis.

SGPT. SGPT concentrations were determined by theTechnicon fluorometric adaptation of Henry's spectrophoto-

metric method. Previous studies have demonstrated rapidalteration in the circulating levels of such enzymes followinginjury to rat livers (6).

Histology. Standard histological procedures were utilized todetermine tissue alterations. In our attempt to determine thepresence of tissue damage with low doses of carcinogen,especially in F/344 female rats, the livers were fixed in 4°

neutral-buffered formalin at pH 7.25 and processed for fatwith sudan red and oil red 0 and with methyl green-pyroninfor RNA. The latter stain has proved to be a sensitivehistological test for cell alteration demonstrating rapid loss ofthe RNA-granular staining pattern. Sections were also fixed incold 100% ethyl alcohol and stained for glycogen by theperiodic acid-Schiff reaction with and without prior diastaseexposure.

RESULTS

a, F Response to a Single Cycle of 2-FAA (Table 1). SDmale and SD female rats received one 3-week exposure to2-FAA at 0.06%, which represents approximately a 0.25carcinogenic dose (24). The rats were bled weekly throughoutthe experiment, which lasted 20 weeks.

Although both groups of rats that were fed 2-FAAdemonstrated elevated a¡F levels after 1 week, only thoselevels achieved by SD female rats were statistically significant(p < 0.01). By the end of the 2nd week, the concentrationsdetermined in both groups were highly significant (p < 0.001 ).The serum concentrations of a,F continued to rise aftercessation of feeding of the carcinogen, reaching a maximalvalue at approximately 28 days and then maintaining aroughly constant level through the 10th week. This levelaveraged 1100 ng/ml for SD male rats (controls, approxi

mately 24 ng/ml) and 644 ng/ml for SD female rats (controls,approximately 39 ng/ml). Both groups of treated ratsdemonstrated normal a\ F levels at the 20th week.

a i F Response to Intermittent and Continuous-Dose Regimen. SD male, and SD female, and F/344 female rats were allused at 125 g. Each group had weight- and sex-paired controls.The dietary regimens used for SD male and SD female ratswere: 0.06% for 2 feeding cycles of 3 weeks each, with 1 weekof normal diet intervening; 0.045, 0.03, or 0.015% fedcontinuously for 42 days. F/344 female rats received only one0.06% feeding cycle or 21 days of each of the lower levels. Therats were bled weekly and followed for 10 weeks (wheresurvivors were available).

Elevation of plasma atF occurred in all SD male rats, thehigher doses producing slightly higher elevations earlier afterfeeding (Table 2). The results obtained with the intermittent0.06% 2-FAA feeding of SD male rats were similar to those ofthe previous experiment when analyzed for statisticallysignificant elevations, but the absolute levels achieved weresomewhat less. The plasma concentration of OL\F becamesignificantly elevated within 1 week and remained elevatedthroughout the feeding regimen. A rough plateau was achievedat 42 days, which was somewhat later than in the previousexperiment, and then remained stable during the experimentalperiod of 70 days. Each of the dietary regimens, whetherintermittant or continuous, evoked a comparable elevation of<*!F in amplitude and timing.

At the termination of the diet, the surviving rats from eachSD male rat dietary regimen were sacrificed and examinedhistologically. The livers of each of the 3 rats of the 0.06%intermittent diet group demonstrated many microscopic,basophilic foci identical to those described in previous reports(4, 10). Two of the 3 survivors of the 0.045% continuousfeeding group demonstrated approximately two-thirds as manyfoci; the other survivor demonstrated only a rare focus ofalteration. Two of 5 rats that had received 0.03% continuousfeeding also demonstrated rare basophilic foci while none ofthe 6 rats that had received 0.015% continuous feedingdemonstrated such foci.

In the female rats, the elevations of OjF produced by alldose regimens were comparable (Tables 3 and 4). However,differences between each of the 3 groups of rats were evident.In general, the elevations of at F demonstrated by the SD

Table 1a, F response to 0.06% 2-FAA for 3 weeks

Experimental rats received 0.06% 2-FAA for 21 days and then normal diet for the remainder of the experiment.

ControlSDmSDmControlSDfSDfNo.

ofrats6565"

Mean ±S.bp<CP<0.0010.01.Mean

a, F levels(ng/ml)Day

747

+80±63

+199+D.3°391535CDay44

+240±52

+252±14644"1242"Day

2137+

6644±33b52+

11346±70bDay

2834

+1001+49

+722±652b624"Day

4228+41042+48"39+

16616±llbDay

7023

+1022±44

+596±430"514bDay24

+566+36

+332+84113"691bDay23

+179+32

+161±982586a57"Day23

+63+3?.

t64±1122176IIeDay

14022

+432+1233+1142

±7

2490 CANCER RESEARCH VOL. 34

on May 14, 2018. © 1974 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

«!-Fetoprotein Elevation by N-2-Fluorenyìacetamide

Table 2a, Fresponse to various dose regimens of 2-FAA in SD male rats

Rats in the 0.06% group received carcinogen-containing diet for two 3-week periods with 1 week of normal diet intervening. Other groupsreceived the diet continuously at the level indicated.

Controls0.06%0.045%0.030%0.015%No.

ofrats56666Day739

±8°91±7b(5)c88±29d56±12d(5)54±6dDay

1425

±120±159±151±75

±95b7b(5)7b6bMean

a,Day

2122

+163±183+178±176

±817b(4)9b3"4bF

levels(ng/ml)Day

2817+

7193±15b(3)298±5b(4)285

±5b182+5bDay

3522

631738735633881b161b

(3)58b63bDay

4229

±409+633+499+408

±1389b180b1lb71bDay

4923+

12451±81b650±80b498±89b411±87b

0 Mean ±S.D.bp< 0.001.c Numbers in parentheses, numbers of survivors.dp<0.01.

Table 3a, F response to various dose regimens of 2-FAA in SD female rats

Rats in the 0.06% group received carcinogen-containing diet for two 3-week periods with 1 week of normal diet intervening.Other groups received the diet continuously at the level indicated.

No.ofControl0.06%0.045%0.03%0.015%rats66666Meana, F levels(ng/ml)Day

763

±13°19±1179

±1178+2688

i 11Day

1443

±1560±7(5)b70±6C59±1250±20Day

2143

±980±7C90±13C89±20C70

±21Day38

+91+90

+112+91

+2891SCtoc36C20eDay44

±474±454±420±397

±355150d154d103d174dDay45

±484+520+673±415±427117d91d25Od113dDay

4937

+6489±92d630+

19d721±91d479

+87d0

Mean ±S.D.Numberincp<0.01.dp<

0.001.parentheses,

number of survivors.

Table 4a,Fresponse to various dose regimens of 2-FAA in Fl344 female rats

Rats in the 0.06% group received carcinogen-containing diet for one 3-week period. Other groups received the dietcontinuously at the level indicated for 21 days.

Control0.06%0.045%0.03%0.015%"

Mean ±S.6p<0.01.cp<

0.001No.

orrats66666D.Mean

a, F levels(ng/ml)Day

759+

17"66±1284±4488±2645±19Day

145876786978714344424Day2163

±680+781

±1470±1181±10Day41

±90±111±107±111±281520b24b19b14bDay49

+426+445±528±507

±3514138C166°107C109eDay

4248

±23508+23C516±25C551±136C510+

86CDay

4946

±1509±IIe579±7C644+

104C508±50C

female rats became statistically significant later than those ofSD male rats (21 days as against 7 days) and earlier than thoseof F/344 female rats (28 days). Both groups of femalesreached maximal elevation at approximately 42 days, whichwas similar to SD male rats, and remained elevated for theremainder of the experiment. It should be emphasized that themajor elevation of »!F in F/344 female rats occurredsubsequent to the cessation of carcinogen administration at 3weeks.

No basophilic foci were observed in any female rats at thetermination of the experiment.

Rat mortality roughly reflected the picture of the histologi-cal findings. Three of 6 SD male rats died during theadministration of 2-FAA in the 0.06% and 0.045% group; 1 of6 died at 0.03%. No male rat died at 0.015%. One of 6 SDfemale rats died at 0.06%. No deaths occurred at any otherdose for SD female rats, and no F/344 female rat died.

«iF Elevations at Low Concentrations of 2-FAA. For

OCTOBER 1974 2491

on May 14, 2018. © 1974 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

F. F. BeckerandS. Sell

determination of a minimal 2-FAA level that would induceatF elevations, SD male rats (6/group) were exposed to verylow doses of carcinogen for 60 days. These diets were preparedby diluting meal containing 0.05% 2-FAA with normal meal ina mechanical mixer. The rats were bled weekly.

With the very low doses of 2-FAA used in this experiment,the elevation of a\ F was directly related to the concentrationof carcinogen in the diet (Table 5). At the end of 1 week, therats receiving 0.005% demonstrated a highly significant elevation of at? (p < 0.001), while the level of rats receiving0.001% was also elevated, but to a lesser degree (p<0.01). Bythe end of the 2nd week, the latter group had also achievedsignificant elevations of a, F (p < 0.001). Those rats receiving0.0005% 2-FAA failed to demonstrate a significant elevationuntil the 28th day (p < 0.01) and progressed to higher levels at35 days of ingestion (p < 0.001). The plasma concentrationsof atF for these 3 groups reached maximum elevationsbetween the 21st and 35th days and remained relativelyconstant through a subsequent 1-month feeding period. Theselevels were related to the dietary concentration of 2-FAA andwere, respectively, 175 ng/ml (0.005%), 115 ng/ml (0.001%),and 98 ng/ml (0.0005%), with controls at 34 ±8 ng/ml.

The rats receiving 0.0001% 2-FAA failed to demonstrateany elevation of ot¡F throughout a 2-month period ofexposure.

Acute Exposure F/344 Female Rats. An attempt was madeto determine the possible influence of cell "injury" asa factor

in plasma a, F elevation by exposing F/344 female rats toeither 0.03% or 0.015% 2-FAA for 3 weeks. These rats wereidentical to those reported in the literature as demonstrating asignificant deficit in hydroxylating capacity (11). Age-pairedrats were used in equal numbers. Three rats were sacrificed pergroup each week for 4 weeks (3 weeks of carcinogen plus 1week of normal diet). The following examinations wereperformed: liver and body weighUo^F and SGPT;histologicalfor fat, glycogen, and RNA patterns (the latter with methylgreen-pyronin stains).

The tt| F elevation was comparable to that described earlierin comparable F/344 female rats, demonstrating a rapidincrease during the 4th week. No elevation of SGPT wasdetected at any time. There was no alteration of liver weightor body weight when compared with matched controls. Nohistológica! alteration was detected at the 0.015% diet level.

The only deviation at 0.03% from normal cytology was asuggestion of a slight fat accumulation in 1 of 3 rats at 3weeks.

DISCUSSION

The major finding of this study was the extremely rapidelevation of the plasma concentration of a¡F after remarkablysmall quantities of the hepatocarcinogen 2-FAA were ingested;these elevations, in many instances, exceeded those associatedwith hepatomas (5,21). These elevated serum a, F concentrations often persisted for a considerable time after intake of thecarcinogen ceased. The use of female rats, which are reportedto have defects in the metabolic activation of 2-FAA (7, 11,12, 16), served to delineate the process of a¡F stimulation. Asderived from the literature, a crude approximation wouldestimate that F/344 female rats possess 10% of the hydroxylating capacity of males (11), while SD female rats aresimilarly defective for arylsulfotransferase activity (7, 11, 12).The SD female rats demonstrated a statistically significantelevation of al F after 2 weeks of exposure to 0.015% 2-FAA,while F/344 female rats demonstrated such elevations after 3weeks. These findings suggest that an extremely small quantityof this carcinogen, or even less of its metabolite(s), is requiredfor activation of at F synthesis. This suggestion was confirmedby a vigorous induction of QI F with brief exposure of SD malerats to dietary concentrations of 0.0005% 2-FAA; this dose isin rough accord with the estimated amount of metabolite(s)required for stimulation as derived from the results in females.The delay in stimulation of atF production in F/344 femalerats suggests that the hydroxy derivative is required for thesequence. If the stimulatory compound was 2-FAA, differences should not have existed between male and female rats inthe rapidity of induction and in the level achieved.

The elevation of plasma at F concentration occurs prior toany detectable histológica! alteration and, in particular, in theabsence of hepatic nodules (10). Most of the regimens usedfailed to produce any significant cellular alteration. The fullysusceptible SD male rats (4, 24) demonstrated highlysignificant increases of u|F at doses and times that were notsufficient to produce tissue alterations. Indeed, a dose of0.005% 2-FAA for 1 week, which produced a significant

Table 5a, F response to low dose levels of 2-FAA in SD male rats

To attain the low levels, diet containing 0.05% 2-1-AAwas diluted to appropriate levels withmeal in a mechanical mixer. The diets were fed continuously. Six rats/group with no deaths.

Mean o, F levels (ng/ml)

Day 7 Day 14 Day 21 Day 28 Day 35

Control0.005%0.001%0.0005%0.0001%38

±8"105 ±19b97

±30e38 ±964

±3038

±7130 ±12b100 ±18b66 ±2835

±343

±19154 ±8b104±28b

84+1940±637

±20159 ±19b107 ±20b90 ±18C60

±3534

±8175 ±12"115 ±8b98±4b45

±21a

Mean ±S.D.bp< 0.001%.cp<0.01%.

2492 CANCER RESEARCH VOL. 34

on May 14, 2018. © 1974 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

t -Fetoprotein Elevation by N-2-Fluorenylacetamide

elevation of «iF, is approximately 1.5% of a calculatedminimal carcinogenic dose (2, 24), 0.075% of an averagecarcinogenic dose, and 5.0% of a minimal dose required toproduce micronodules.

Among the mechanisms that have been suggested to resultin ttiF induction are hepatocyte cell division (1, 20) and cellinjury (8). These associations, however, could not be causallyrelated to the elevations noted in the present experiments. Ithas been reported by others that no cell division was detectedin livers of F/344 female rats at doses of 0.015% 2-FAA (12).Further, in parallel experiments, we have been unable todetect any significant alteration in DNA syntheses in SDfemale rats at this dose or in SD male rats at a dose of 0.005%during the 3 weeks of exposure. The failure to demonstrateany form of detectable histological alteration in F/344 femalerats fed 0.015% to 0.03% 2-FAA, the lack of alteration inSGPT levels, and a normal liver and body weight all militateagainst cell injury as a causal factor. Almost all toxins thathave been reported to excite a, F production do so throughcell necrosis and compensatory mitosis (1,21). The persistenceof a¡F elevations for at least 1 month following a 3-weekexposure of F/344 female rats to 0.015% also makes injury anunlikely stimulus because the elevation that follows injury isusually transient.

We cannot define the mechanism that is responsible for thepersistence of the elevated o^F levels. Whether this representspersistence of 2-FAA or its metabolites in complex withchromatin (9) or the turnover rate of "activated" hepatocytes

is unclear. On the basis of these data, it is our hypothesis thata metabolite of 2-FAA interacts in a highly selective mannerwith the genetic locus responsible for the control of cti Fsynthesis. This complex may be remarkably stable resulting instimulation of at F synthesis for a protracted period in theabsence of continued exposure. The height of the elevationachieved at a given dietary concentration is the result of anequilibrium between the rate at which metabolic activation of2-FAA occurs, the rate of complex formation at crucialgenetic loci, and the ability of the cell to deactivate 2-FAAmetabolically whether in macromolecular complex or "free."

The rough sigmoidal quality of the curves of al F induction andthe variability of the height of the final "plateaus" suggest that

more than 1 site for o^F stimulation might exist. It appearsprobable that the interaction between 2-FAA and thehepatocyte which results in c^F stimulation is but one of aseries of such macromolecular interactions required forcarcinogenesis. Indeed, this initial response may be requiredfor a relatively transient period. The lack of necessity for apermanent complex at this site could account for the findingof hepatomas that produce little or no at F (5).

REFERENCES

1. Abelev, G. I. Alpha-Fetoprotein in Oncogenesis and Its Associationwith Malignant Tumors. Advan. Cancer Res., 14: 295-358, 1971.

2. Albert, R. E., Burns, F. J., Bilger, L., Gardner, D., and Troll, W.Cell Loss and Proliferation Induced by W-2-Fluorenylacetamide inthe Rat Liver in Relation to Hepatoma Induction. Cancer Res., 32:2172-2177, 1972.

3. Alpert, M. E., Uriel, J., and de Néchaud,B. Alpha-Fetoprotein inthe Diagnosis of Human Hepatomas. New Engl. J. Med., 278:984-986, 1968.

4. Becker, F. F., and Klein, K. M. The Effect of L-Asparaginase onMitotic Activity during jV-Fluorenylacetamide Hepatocarcino-genesis: Sub-populations of Nodule Cells. Cancer Res., 31:169-173, 1971.

5. Becker, F. F., Klein, K. M., Wolman, S. R., Asofsky, R., and Sell,S. Characterization of Primary Hepatocellular Carcinomas andInitial Transplant Generations. Cancer Res., 33: 3330-3338, 1973.

6. Becker, F. F., Williams, R. B., Jr., and Voogd, J. L. Effect ofX-Irradiation in Sub-lethal to Supra-lethal Dosage on SerumGlutamic OxaloaceticTransaminase. Radiation Res., 20: 221-226,

1963.7. DeBaun, J. R., Miller, E. C. and Miller, J. A. ¿V-Hydroxy-2-acetyl-

aminofluorene Sulfotransferase: Its Probable Role in Carcinogenesis and in Protein-(methion->S-yl) Binding in Rat Liver. CancerRes., 30: 577-595, 1970.

8. de Néchaud,B., and Uriel, J. AntigènesCellulaires Transitoires duFoie de Rat. III. Mode de Réapparitionde l'a-Foetoprotéine aucours de l'Hcpatocarcinogcnèse Chimique. Intern. J. Cancer, 77:

104-115, 1973.9. Epstein, S. M., McNary, J., Bartus, B., and Farber, E. Chemical

Carcinogenesis: Persistence of Bound Forms of 2-Fluorenyl-acetamide. Science, 762: 907-908, 1968.

10. Farber, E. Similarities in the Sequence of Early Histologie ChangesInduced in the Liver of the Rat by Ethionine, 2-Acetylamino-fluorene, and 3<-Methyl-4-dimethylaminoazobenzene. Cancer Res.,76: 142-148, 1956.

11. Gutmann, H. R., Malejka-Giganti, D., Barry, E. J., and Rydell, R.E. On the Correlation between the Hepatocarcinogenicity of theCarcinogen, ./V-2-Fluroenylacetamide, and Its Metabolic Activationof the Rat. Cancer Res., 32: 1554-1561, 1972.

12. Jackson, C. D., and Irving, C. C. Sex Differences in CellProliferation and TV-Hydroxy-2-acetylaminofluorene Sulfotransferase Levels in Rat Liver during 2-Acetylaminofluorene Administration. Cancer Res.,^2: 1590-1594, 1972.

13. Kitagawa, T., Yokochi, T., and Sugano, H. a-Fetoprotein andHepatocarcinogenesis in Rats Fed 3'-Methyl-4-(dimethylamino)

Azobenzene or jV-2-Fluorenylacetamide. Intern, J. Cancer, 70:368-381, 1972.

14. Kroes, R., Williams, G. M., and Weisburger, J. H. Early Appearanceof Serum a-Fetoprotein during Hepatocarcinogenesis as a Functionof Age of Rats and Extent of Treatment with 3'-Methyl-4-di-

methylaminoazobenzene. Cancer Res., 32: 1526-1532, 1972.15. Kroes, R., Williams, G. M., and Weisburger, J. H. Early Appearance

of Serum a-Fetoprotein as a Function of Dosage of VariousHepatocarcinogens. Cancer Res., 33: 613-617, 1973.

16. Miller, J. A. Carcinogenesis by Chemicals: An Overview-G. H. A.Clowes Memorial Lecture. Cancer Res., 30: 559-576, 1973.

17. Monjour, L., and Mariage, C. Antigènes Embryonnaires etAflutoxine. Compt. Rend. Soc. Biol., 76.?: 1288-1295, 1969.

18. Sell, S. Radioimmunoassay of Rat Alpha Fetoprotein (a, F).Cancer Res., 33: 1010-1015, 1973.

19. Sell, S., and Gord, D. Rat Alpha-Fetoprotein. III. Refinement ofRadioimmunoassay for Detection of 1 ng Rat a, F. Immuno-chemistry, 10: 439-442, 1973.

20. Sell, S., Nichols, M., Becker, F. F., and Leffert, H. Rat a, F. X.Hepatocyte Proliferation and a,-Fetoprotein in Pregnant,Neonatal, and Partially Hepatectomized Rats. Cancer Res., 34:865-871, 1974.

21. Sell, S., and Wepsic, H. T. Alpha-Fetoprotein. In: F. F. Becker,(ed.), The Liver-Mechanisms of Its Function and Malfunction.New York: Marcel Dekker, Inc., in press.

22. Stanislawski-Birencwajg, M., Uriel, J., and Grabar, P. Association of

OCTOBER 1974 2493

on May 14, 2018. © 1974 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

F. F. Becker and S. Sell

Embryonic Antigens with Experimentally Induced Hepatic Lesions and Their Relationship to Hepatocarcinogenesis. Cancer Res., 31:in the Rat. Cancer Res., 27: 1990-1997,1967. 1-3,1971.

23. Tatarinov, J. S. New Data on the Embryo-Specific Antigenic 25. Uriel, J. Transitory Liver Antigens and Primary Hepatoma in ManComponents of Human Blood Serum. Vopr. Med. Khim., JO: and Rat. Pathol. Biol. Semaine Hop., 17: 877-884, 1969.584-589, 1964. 26. Watabe, H. Early Appearance of Embryonic -y-Globulin in Rat

24. Teebor, G. W., and Becker, P. R. Regression and Persistence of Serum during Carcinogenesis with 4-Dimethylaminoazobenzene.Hyperplastic Hepatic Nodules Induced by W-2-Fluorenylacetamide Cancer Res., 31: 1192-1194, 1971.

2494 CANCER RESEARCH VOL. 34

on May 14, 2018. © 1974 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

1974;34:2489-2494. Cancer Res   Frederick F. Becker and Stewart Sell  Hepatocarcinogenesis

-2-FluorenylacetamideN-Fetoprotein in 1αEarly Elevation of

  Updated version

  http://cancerres.aacrjournals.org/content/34/10/2489

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

  .pubs@aacr.orgDepartment at

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/34/10/2489To request permission to re-use all or part of this article, use this link

on May 14, 2018. © 1974 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from