Embed Size (px)
Citation preview
[CANCER RESEARCH 41, 1011-1014. March 1981]0008-5472/81 70041-OOOOS02.00
Aflatoxicol-induced Hepatocellular Carcinoma in Rainbow Trout (Salmo
gairdneri) and the Synergistic Effects of Cyclopropenoid FattyAcids1
Grant L. Schoenhard,2 Jerry D. Hendricks,3 Joseph E. Nixon, Donald J. Lee,4 Joseph H. Wales, Russell O.
Sinnhuber, and Norman E. Pawlowski
Department of Food Science and Technology, Oregon State University, Corvallis. Oregon 97331
ABSTRACT
Aflatoxicol (AFL), a major metabolite of aflatoxin B, (AFB,) inthe Mt. Shasta rainbow trout (Salmo gairdneri), was found toproduce hepatocellular carcinoma in trout. It was administeredin a casein diet to duplicate groups of 120 fingerling trout. Inthe same manner, additional duplicate groups received one ofthe following: no toxicant; AFB,; the diastereomer of AFL(AFL1); cyclopropenoid fatty acids (CPFA); and CPFA plusAFBi, AFL, and AFL'. Eight months after the initiation of the
study, the following incidences of carcinoma were observed:control (0%); 20 ppb AFB, (56%); 29 ppb AFL (26%); 61 ppbAFL' (0%); 50 ppm CPFA (3%); 20 ppb AFB, plus 50 ppm
CPFA (96%); 29 ppb AFL plus 50 ppm CPFA (94%); and 61ppb AFL' plus 50 ppm CPFA (55%), showing both the carci-
nogenicity of AFL and the synergistic effects of CPFA. Twelvemonth incidences were correspondingly higher in all cases.Aflatoxin M,, another metabolite of AFB, in rainbow trout, wasreported previously to be carcinogenic in trout. These resultssupport the hypothesis that metabolism in rainbow trout doesnot effectively detoxify AFB,, but rather the formation of AFLextends the carcinogenicity of AFB, and may contribute to thehigh sensitivity of rainbow trout to AFB,.
INTRODUCTION
AFL5 is formed in vitro during the incubation of AFB, with
postmitochondrial fractions prepared from chickens, ducks,turkeys, rabbits (14, 18), trout (18, 21, 22), monkeys, andhumans (18). The mouse, rat, and guinea pig apparently produced no (15) or very little AFL (18). The species which convertAFB, to AFL are capable of reconverting AFL to AFB, (1 2, 15,18).
It is well documented that AFB, is carcinogenic and requiresmetabolic activation to elicit many of its biological effects (1,3-6, 19, 20, 28-31). AFL is toxic to Bacillus subtilis after
activation with rainbow trout microsomes (21, 22), brings about
' This is Technical Paper 4768 of the Oregon Agricultural Experiment Station.
This investigation was supported by USPHS Training Grant FD 00010 andResearch Grants ES 00256, ES 00210. ES 01926, ES 00550, and ES 00541 ofthe United States Department of Health, Education and Welfare.
2 Present address: Department of Drug Metabolism and Radiochemistry,
Searle Laboratories, Box 5110, Chicago, III. 60680. The material presentedherein is taken in part from a dissertation submitted in partial fulfillment of therequirements for the Ph.D. in 1974.
3 To whom requests for reprints should be addressed.4 Present address: Agricultural Research Center, Washington State University,
Pullman, Wash. 99163.5 The abbreviations used are: AFL, aflatoxicol; AFB,, aflatoxin B,; AFMlf
aflatoxin M,: AFL', the diastereomer of aflatoxicol; CPFA, cyclopropenoid fatty
acid; AFQ,, aflatoxin Q,.Received February 6, 1978; accepted November 21, 1980.
unscheduled DNA synthesis in fibroblasts incubated with a ratliver postmitochondrial supernatant fraction (26), and is aframe-shift mutagen to Salmonella typhimurium in the presenceof a rat postmitochondrial liver fraction (13, 34, 35).
In rainbow trout, AFL is a major metabolite. In addition, AFM,is also found. The latter is a carcinogen in trout (25) and rats(33). It was hypothesized that AFL would also produce tumors.The objective of the study described in this paper was toevaluate this hypothesis for AFL as well as its diastereomer,AFL', which is formed during the chemical reduction of AFB,
to AFL (16, 21).Previous experiments have demonstrated the strong syner
gistic effects of CPFA on AFB,, AFM,, and AFQ, carcinogen-esis in rainbow trout (8-10, 25) and the primary carcinogenicity
of CPFA to trout (7, 8, 24). We were therefore interested indetermining the possible synergism of CPFA in combinationwith AFL.
MATERIALS AND METHODS
Experimental Animals and Diets. Mt. Shasta rainbow trout(Salmo gairdneri} eggs were spawned, hatched in our laboratory facility, and the fry fed a purified casein diet (21) for 4months prior to initiation of the feeding trial. For this study,groups of 120 fingerlings were randomly selected. Each groupwas restrained in a 6-gallon plastic bucket with many holes for
water flow and an effective rearing area of 0.5 cu ft. Twobuckets for duplicate diets were suspended in 4-ft-diameter
fiberglass tanks. The water flow rate in the tanks was 4 gallons/min at a temperature of 12°,with an oxygen content of 8.5 to
9.5 ppm. After the first 2 months of the study, each bucket oftrout was transferred to an individual tank.
Chemicals. AFB, was purchased from Calbiochem-Behring
Corp., La Jolla, Calif., and the purity was determined to begreater than 99%, as described previously (23). AFL and AFL'
were prepared by chemical reduction of AFB, (16, 21). TheCPFA were prepared as reported previously (10). Tricainemethanesulfonate was purchased from Endo Products, Inc.,Hanover, N. J.
Treatment. Duplicate groups of trout received the caseindiet with the toxicants and dose levels indicated in Table 1.One ml of ethanol per 100 g of herring oil in the diet was usedas the toxicant carrier and was added to the control diets. Thelevels of AFL and AFL' incorporated in the diets were calculated
based on the literature values for the extinction coefficientsreported in the literature (2). During the course of the study,sufficient AFL and AFL' were synthesized to reevaluate theextinction coefficients. The levels of AFL and AFL' reported in
Table 1 were determined from the results of the réévaluationof
MARCH 1981 1011
Research. on January 17, 2021. © 1981 American Association for Cancercancerres.aacrjournals.org Downloaded from
G. L. Schoenhard et al.
Table 1
Incidence of hepatocellular carcinoma in the Mt. Shasta rainbow trout (S. gaìrdneri)
Duplicate tanks of trout were fed a purified diet (17) containing the toxicants. Trout were randomlyselected and sacrificed at 4-month intervals of a 12-month period.
Toxicant indietNone
AFB, (20 ppb)AFL (29 ppb)AFL' (61 ppb)
CPFA (50 ppm)AFB, (20 ppb) + CPFA (50 ppm)AFL (29 ppb) + CPFA (50 ppm)AFL' (61 ppb) + CPFA (50 ppm)4-mo.
sample,
duplicate (carcinoma/40trout)0
00006320
0000321(Of
(0)(0)(0)
(0)(11)(6)(4)8-mo.
sample, duplicate (carci
noma/40trout)0
1811
01
3938250
2710
01
393825(0)
(56)(26)
(0)(3)
(96)(94)(55)12-mo.
sample, duplicate(carcinoma/sample)0/38
29/3725/31
7/3616/3832/3228/2831/350/38
33/3821/2610/3413/3632/3235/3537/37(0)(83)
(81)(24)(39)
(100)(100)
(94)1Numbers in parentheses, percentage.
the extinction coefficients (16).Termination of Experiment. Four and 8 months following
initiation of the study, 40 trout were randomly selected fromeach tank. The remaining trout were sacrificed after 12 months.The trout were killed with tricaine methanesulfonate, weighed,and autopsied for gross abnormalities. The livers and kidneyswere examined for tumors and toxic damage. They were preserved in Bouin's fixative. These tissues were sectioned at 4
fim and stained with hematoxylin and eosin for detailed examination.
RESULTS
The results of administration of AFBi, AFL, and AFL' with
and without CPFA are given in Table 1. They clearly demonstrate that AFL produced hepatocellular carcinoma (Figure 1)in rainbow trout. The relative potency of the toxicants was mostevident at 8 months, indicating that AFL was less carcinogenicthan AFB, by 50% or more. This relative difference was maskedat 12 months, since the AFL carcinogenic response caught upwith the AFB, response that was near maximum expression.Direct comparison of the relative potency of the aflatoxins wasnot possible because the dietary level of AFL was higher thanAFB, and because AFL' was higher than AFL (see "Materialsand Methods" for explanation).
Hepatocellular carcinomas were also observed in trout fedCPFA in the diet, confirming the carcinogenicity of CPFA totrout as described previously by Hendricks ef al. (7, 8) andSinnhuber ef al. (24). The addition of CPFA to diets containingAFB,, AFL, and AFL' also enhanced the incidence of carci
noma. The synergistic effect of CPFA was clearly seen at 8months but masked at 12 months due to high tumor incidencesin the aflatoxin diets. Sterculic acid, a component of CPFA,and/or its methyl ester, methyl sterculate, have been shownpreviously to enhance AFB,-, AFM,-, and AFQ,-induced tumorgrowth (8-10, 25).
In addition to carcinoma, chronic toxicity was evident in thelivers of the trout fed AFB,, AFL, and AFL'. Hypertrophy of
parenchymal cells was common, resulting in swollen hydropiccells with enlarged pleomorphic, hyperchromatic nuclei. Thesecells became degenerate and necrotic, and regenerative activity was common in these livers. AFB, caused greater toxicinjury than did AFL (Figs. 2 to 4), and the diastereomer AFL'
was much less toxic than either AFB, or AFL.The addition of CPFA to the diets resulted in more severe
toxic effects. Typical CPFA-induced pathology, i.e., cellularand nuclear hypertrophy, aberrant endoplasmic reticulum (cy-
toplasmic striations), and fibrosis (27), combined with aflatoxinlesions, produced livers with many degenerate and necroticcells and abundant regenerative activity. No aflatoxin-related
effects were seen in the kidneys.
DISCUSSION
The carcinogenicity we have found for AFL may be a resultof its direct activation to the ultimate carcinogen. Alternatively,AFL may be converted back to AFB,, as Loveland ef al. (12)reported, and the AFB,, not AFL, may be activated. The enzymesystem which converts AFL to AFB, is found primarily in themicrosomal fraction, requires a hydrogen acceptor such asNADP, is not dependent on glucose-6-phosphate or glucose-6-phosphate dehydrogenase, and is not inhibited by carbonmonoxide (11, 18). Under normal physiological conditions, theratio of cellular NADPH/NADP would be greater than 1 andwould favor maintenance of AFL in preference to conversion toAFB,. The higher ratio of NADPH would favor microsomalmonooxygenase activation of either AFB, or AFL to an ultimatecarcinogenic form (4).
It is not clear why AFL', the unnatural isomer, was carcino
genic, since Schoenhard ef al. (21) showed previously thatAFL' was not activated by trout postmitochondrial fraction to a
metabolite toxic to B. subtilis. It may be first converted to AFB,and then activated to the ultimate carcinogen. An inefficientconversion to AFB, may explain the low carcinogenicity. Alternatively, AFL' may be directly activated, and its lower carci
nogenicity may be due to lack of efficiency of this activation orto hindered binding at the target due to stereospecificity.
Efforts have been made to explain the differences in toxicityand carcinogenicity of AFB, and AFL among species based onvariations in in vitro metabolism. Roebuck and Wogan (17)reported that no consistent pattern was correlated to the invitro metabolism of AFB,. On the other hand, Salhab andEdwards (18) noted that trout and rabbit preparations weremost active in the reduction of AFB, to AFL. They suggestedfurther that species that were very sensitive to AFB, had ahigher ratio of AFB, reductase activity to AFL dehydrogenaseactivity. The opposite was true for the less sensitive species.The rainbow trout appears to be the most sensitive species toAFB,-induced carcinogenicity. Further, AFM,, another of the
AFB, metabolites (12) in rainbow trout, is also carcinogenic;the metabolic conversion of AFB, to AFM, is a nonreversiblereaction. Thus, carcinogenicity of AFM, cannot be explainedby metabolic conversion to AFB,, which lends support to directactivation of AFB, metabolites to ultimate carcinogenic metab-
1012 CANCER RESEARCH VOL. 41
Research. on January 17, 2021. © 1981 American Association for Cancercancerres.aacrjournals.org Downloaded from
AFL-induced Carcinoma in Trout
olites. In this paper, we report that AFL, the major metabolitein trout, is carcinogenic. We postulate that the extreme sensitivity of rainbow trout is due in part to the tact that, in additionto AFB,, its major metabolites are also carcinogenic. For thisspecies, the pattern of in vitro metabolism appears to beconsistent with its susceptibility to aflatoxin-induced hepato-
cellular carcinoma.
ACKNOWLEDGMENTS
We thank Richard A. Foster, Jr.. George B. Putnam, Samuel E. Howell, L. J.Hunter, and Theodore Will tor their technical assistance.
REFERENCES
1. Campbell, T. C., and Hayes, J. R. The role of aflatoxin metabolism in itstoxic lesion. Toxicol. Appi. Pharmacol., 35. 199-222, 1976.
2. Cole, R. J., Kirksey, J. W., and Blankenship, B. R. Conversion of aflatoxinB, to isomerie hydroxy compounds by Rhizopus spp. J. Agrie. Food Chem.,20: 1100-1102, 1972.
3. Garner, R. C. Reduction of binding of [14C]aflatoxin Bi to rat liver macro-
molecules by phenobarbitone pretreatment. Biochem. Pharmacol., 24:1553-1556. 1975.
4. Garner, R. C., Miller, E. C., and Miller. J. A. Liver microsomal metabolism ofaflatoxin Bt to a reactive derivative toxic to Salmonella typhimurium TA1530. Cancer Res., 32: 2058-2066, 1972.
5. Garner, R. C., Miller. E. C., Miller, J. A., Garner, J. V., and Hansen, R. S.Formation of a factor lethal for S typhimurium TA 1530 and TA 1531 onincubation of aflatoxin Bi with rat liver microsomes. Biochem. Biophys. Res.Commun., 45: 774-780, 1971.
6. Garner, R. C., and Wright, C. M. Binding of [!4C]aflatoxin B, to cellular
macromolecules in the rat and hamster. Chem.-Biol. Interact., 71: 123-131,1975.
7. Hendricks. J. D., Sinnhuber, R. O., Loveland, P. M., Pawlowski, N. E., andNixon, J. E. Hepatocarcinogenicity of glandless cottonseeds and cottonseedoil to rainbow trout (Salmo gairdneri). Science (Wash. D. C.), 208:309-311,1980.
8. Hendricks, J. D., Sinnhuber, R. O., Nixon, J. E., Wales, J. H., Masri, M. S..and Hsieh, D. P. H. Carcinogenic response of rainbow trout (Sa/mo gairdneri)to aflatoxin Q, and synergistic effect of cyclopropenoid fatty acids. J. Nati.Cancer Inst., 64: 523-527, 1980.
9. Lee, D. J., Wales, J. H., Ayres, J. L., and Sinnhuber, R. O. Synergismbetween cyclopropenoid fatty acids and chemical carcinogens in rainbowtrout (Salmo gairdneri). Cancer Res., 28: 2312-2318, 1968.
10. Lee, D. J., Wales. J. H., and Sinnhuber, R. O. Promotion of aflatoxin-inducedhepatoma growth in trout by methyl malvalate and sterculate. Cancer Res.,31: 960-963. 1971.
11. Loveland, P. M., Nixon, J. E.. Pawlowski, N. E.. Eisele, T. A.. Libbey, L. M..and Sinnhuber, R. O. Aflatoxin Bi and aflatoxicol metabolism in rainbowtrout (Sa/mo gairdneri) and the effects of dietary cyclopropene. J. Environ.Pathol. Toxicol.. 2: 707-718, 1979.
12. Loveland, P. M., Sinnhuber, R. O., Berggren, K. E., Libbey, L. M., Nixon, J.E., and Pawlowski, N. E. Formation of aflatoxin B, from aflatoxicol byrainbow trout (Salmo gairdneri) liver in vitro. Res. Commun. Chem. Pathol.Pharmacol., i6: 167-170, 1977.
13. McCann, J., Choi, E., Yamasaki, E., and Ames, B. N. Detection of carcinogens and mutagens in the Salmonella microsome test: assay of 300 chemicals. Proc. Nati. Acad. Sei. U. S. A., 72: 5135-5139, 1975.
14. Patterson, D. S. P., and Roberts, B. A. The in vitro reduction of aflatoxins B,
and Bj by soluble avian liver enzymes. Food Cosmet. Toxicol.. 9 829-837.1971.
15. Patterson, D. S. P., and Roberts, B. A. Aflatoxin metabolism in duck-liverhomogenates: the relative importance of reversible cyclopentenone reduction and hemiacetal formation. Food Cosmet. Toxicol.. 10: 501-512, 1972.
16. Pawlowski, N. E., Schoenhard, G. L., Lee, D. J., Libbey. L. M., Loveland, P.M.. and Sinnhuber, R. O. Reduction of aflatoxin B, to aflatoxicol. J. Agrie.Food Chem., 25: 437-438, 1977.
17. Roebuck, B. D., and Wogan, G. N. Species comparison of in vitro metabolismof aflatoxin B,. Cancer Res., 37: 1649-1656, 1977.
18. Salhab, A. S., and Edwards, G. S. Comparative in vitro metabolism ofaflatoxicol by liver preparations from animals and humans. Cancer Res., 371016-1021. 1977.
19. Schoenhard, G. L.. Bishop, P. E., and Lee, D. J. Aflatoxin B, transformationby rainbow trout (Salmo gairdneri) to a product(s) lethal to Bacillus subtilisGSY 1057. Fed. Proc., 32: 277, 1973.
20. Schoenhard, G. L., Bishop, P. E., Lee, D. J., and Sinnhuber, R O Bacillussubtilis GSY 1057 assay for aflatoxin B, activation by rainbow trout (Salmogairdneri). J. Assoc. Off Anal. Chem., 58: 1074-1076, 1975.
21. Schoenhard, G. L., Lee. D. J., Howell, S. E., Pawlowski, N. E., Libbey. L.M., and Sinnhuber, R. O. Aflatoxin B, metabolism to aflatoxicol and derivatives lethal to Bacillus subtilis GSY 1057 by rainbow trout (Salmo gairdneri)liver. Cancer Res., 36: 2040-2045, 1976.
22. Schoenhard, G. L., Lee, D. J., and Sinnhuber, R. O. Aflatoxin Bi activationand aflatoxicol toxicity in rainbow trout (Salmo gairdneri). Fed. Proc., 33:247, 1974.
23. Schoenhard, G. L., Sinnhuber, R. O., and Lee, D. J. Preparation of '"C-
labeled aflatoxin B,. J. Assoc. Off. Anal. Chem., 56 643-646. 1973.24. Sinnhuber, R. O., Hendricks, J. D., Putnam, G. B., Wales. J. H.. Pawlowski,
N. E., Nixon, J. E.. and Lee. D. J. Sterculic acid, a naturally occurringcyclopropene fatty acid, a liver carcinogen to rainbow trout. Fed. Proc , 35505. 1976.
25. Sinnhuber. R. O., Lee, D. J., Wales, J. H.. Landers. M. K.. and Keyl. A. C.Hepatic carcinogenesis of aflatoxin M, in trout (Salmo gairdneri) and itsenhancement by cyclopropene fatty acids. J. Nati. Cancer Inst.. 53 1285-1288, 1974.
26. Stich, H. F., and Laishes, B. A. The response of xeroderma pigmentosumcells and controls to the activated mycotoxins, aflatoxins, and sterigmato-cystin. Int. J. Cancer. 16: 266-274, 1975.
27. Struthers, B. J., Wales, J. H., Lee. D. J.. and Sinnhuber, R. O. Livercomposition and histology of rainbow trout fed cyclopropenoid fatty acids.Exp. Mol. Pathol., 23: 164-170, 1975.
28. Swenson, D. H., Lin, J-K., Miller, E. C., and Miller. J. A. Aflatoxin B.-2.3-oxide as a probable intermediate in the covalent binding of aflatoxins Bi andB2 to rat liver DNA and ribosomal RNA in vivo. Cancer Res.. 37: 172-181,1977.
29. Swenson, D. H., Miller, E. C., and Miller. J. A Aflatoxin B,-2,3-oxide:evidence for its formation in rat liver in vivo and by human liver microsomesin vitro. Biochem. Biophys. Res. Commun., 60: 1036-1043, 1974.
30. Swenson, D. H., Miller, J. A., and Miller, E. C. 2,3-Dihydro-2,3-dihydroxy-aflatoxin B,: an acid hydrolysis product of an RNA-aflatoxin B, adduciformed by hamster and rat liver microsomes in vitro. Biochem. Biophys. Res.Commun., 53: 1260-1267, 1973.
31. Swenson, D. H.. Miller, J. A., and Miller, E. C. The reactivity and carcino-genicity of aflatoxin B,-2,3-dichloride, a model for the putative 2,3-oxidemetabolite of aflatoxin B,. Cancer Res., 35: 3811-3823, 1975.
32. Wogan, G. N. Aflatoxin carcinogenesis. Methods Cancer Res., 7 309-344,1973.
33. Wogan, G. N.. and Paglialunga. S. Carcinogenicity of synthetic aflatoxin M,in rats. Food Cosmet. Toxicol., 12: 381-384, 1974.
34. Wong, J. J., and Hsieh D. P. H. Mutagenicity of aflatoxins related to theirmetabolism and carcinogenic potential. Proc. Nati. Acad. Sei. U. S. A., 73:2241-2244. 1976.
35. Wong, J. J., Singh. R.. and Hsieh, D. P. H. Mutagenicity of fungal metabolitesrelated to aflatoxin synthesis. Mutât.Res . 44 447-450. 1977.
Fig. 1. A portion of a trabecular hepatocellular carcinoma initiated in a trout fed 29 ppb AFL for 12 months. Note the extreme basophilia, enlarged nuclei, andmitotic figures in the tumorous cells and the normal tissue. H & E, x 512.
Fig. 2. Normal rainbow trout liver tissue from a control trout sampled at 6 months. Note the small uniform size of both the cells and nuclei and the normal 2-cell-wide hepatic cords. H & E, x 512.
Fig. 3. Liver tissue from a rainbow trout fed 20 ppb AFB, for 6 months. Note the hypertrophy of individual hepatocytes and extreme nuclear aberrations. Abundantregenerative activity (foci of basophilic cells) and disruption of normal liver structure are evident. H & E. x 512.
Fig. 4. Liver tissue from a rainbow trout fed 29 ppb AFL for 6 months. Cellular hypertrophy is evident but not as severe as in Fig. 3. Nuclear aberrations arecommon, but regenerative activity is absent. H x E, its 512.
MARCH 1981 1013
Research. on January 17, 2021. © 1981 American Association for Cancercancerres.aacrjournals.org Downloaded from
G. L. Schoenhard et al.
SÃ
1 Af-^PlLt^.
*•i ^^
*V^ a% ¥$rV« ¿r^** * r2'ÕfeA^te^NZ-
^^1KSÃ*&^^^^'^/S^^
1014 CANCER RESEARCH VOL. 41
Research. on January 17, 2021. © 1981 American Association for Cancercancerres.aacrjournals.org Downloaded from
1981;41:1011-1014. Cancer Res Grant L. Schoenhard, Jerry D. Hendricks, Joseph E. Nixon, et al. Cyclopropenoid Fatty Acids
) and the Synergistic Effects ofSalmo gairdneri(Aflatoxicol-induced Hepatocellular Carcinoma in Rainbow Trout
Updated version
http://cancerres.aacrjournals.org/content/41/3/1011
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/41/3/1011To request permission to re-use all or part of this article, use this link
Research. on January 17, 2021. © 1981 American Association for Cancercancerres.aacrjournals.org Downloaded from
