The Influence of a Low Lipotrope Diet on Response of ... · 3Chemicals were supplied by Dr. C. C....

[CANCER RESEARCH 28, 2327-2337, NOVEMBER 1968]

The Influence of a Low Lipotrope Diet on Response of Maternal andFetal Rats to Lasiocarpine1 1.2

P. M. Newberne

Department of Nutrition and Food Science, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139

SUMMARY

Lasiocarpine has been administered intragastrically topregnant rats maintained on control and on lipotrope-deficientdiets during gestation. Effects of the toxin on the maternalliver have been noted as well as the transplacental effects onthe newborn rat at birth and at seven weeks postpartum. Aschedule of treatment was achieved which resulted in nodetectable morphologic alterations in the maternal liver butcaused fetal liver necrosis. The low lipotrope diet enhanced theeffects of lasiocarpine on both maternal and fetal liver.Exposure to the toxin in utero resulted in significant liverlesions in seven-week-old rats with most severe changesobserved in offspring littered to females fed the low lipotropediet.

INTRODUCTION

The pyrrolizidine alkaloids occur in many species of threeimportant plant families, including the genera Senecio,Crotalaria, and Heliotropium, and they constitute a hazard toman and animals in many parts of the world. Sheep, cattle,and horses develop a slowly progressive fatal liver disease afteringesting the toxic plants (2, 3). Cirrhosis of the liver in horsesas a result of feeding on ragwort (Senecio jacobea) was reported as early as 1903 (2), and according to Campbell (4)this was the first indication that this plant was hepatotoxic.Reports from widely separated areas of the world, includingNew Zealand (Winton disease), Germany (Schweinsberger's

disease), Nova Scotia (Pictou disease), Norway (Sirasyke),Nebraska (Walking disease), and South Africa (Molten diseaseor Dunsiekte) have confirmed the extensive distribution ofliver disease resulting from ingestion of various species ofSenecio (9).

Contamination of edible flour with Senecio species has beenassociated with human liver disease in Cape Province and inCape Town (17). Potentially dangerous consequences may alsooccur from the administration of the alkaloids in medicinalpreparations (13), and a disease in children has been associated

'This study was supported in part by NIH Research Grants ES-00183and CA-00870.

2This manuscript is Contribution No. 1300 from the Department of

Nutrition and Food Science, Massachusetts Institute of Technology.3Chemicals were supplied by Dr. C. C. J. Culvenor, Commonwealth

Scientific and Industrial Research Organization, Melbourne, Australia.

with consumption of "bush tea" contaminated with Senecio

alkaloids in the West Indies and in South Africa (1, 9). Thecommon occurrence in man of cirrhosis and primary livercancer in areas where Senecio is known to be ingested hassuggested an etiologic association which thus far is unproven.

A chronic, progressive liver disease has been induced in ratsby administration of a single dose of a pyrrolizidine alkaloid(14). It is significant that the toxicity of the alkaloid wasaccelerated when dietary protein was restricted. In addition,malignant liver cell tumors have been induced in rats withmoderate, intermittent doses of pyrrolizidine alkaloids H 5)and Campbell (4) has produced tumors in fowls byadministering an alkaloid extracted from Senecio jacobea(seneciphylline).

The preceding observations point to a direct action by theSenecio alkaloids on the liver parenchymal cells and illustratethe potential carcinogenic hazards to man and animals. Wehave been interested for a number of years in the relationshipof dietary factors to the induction of liver cirrhosis andcarcinoma. This paper reports the results of investigations inwhich pregnant and lactating female rats were fed control dietsor diets low in lipotropes and exposed to lasiocarpine.

MATERIALS AND METHODS

Female rats of the Charles River caesarean-derived strain(Charles River Breeding Laboratories, North Wilmington,Massachusetts) were fed their respective diets beginning onDay one of pregnancy, based on positive vaginal swabs, andcontinued on diet through lactation. Composition of the dietsused in the experiments is shown in Table 1. The peanut mealused in the diet was alcohol extracted and shown to be free ofaflatoxin by chemical assay. Single, screen-bottom cages wereused to house the pregnant females; feed and water weresupplied ad libitum. Lasiocarpine solutions3 as the sulfate

were freshly preparedDosing was by gastric intubation. Some females were given asingle dose of lasiocarpine on Day 13 and were killed on Day20. Others were dosed on Day 14 and killed on Day 21, andstill others were dosed on two gestational days (13 and 17)and killed on Day 20. Some females from the various groupswere allowed to litter normally and, where possible, some ofthe offspring were continued on diet to weaning and beyondin order to observe later postnatal effects. Gross observationswere made, placenta! and fetal weights were recorded, andtissues were taken from all specimens and routinely processedfor histologie study.

NOVEMBER 1968 2327

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P. M. Newberne

Table 1

IngredientsCasein

(Alcoholic extract)Peanut meal (Alcoholic extract)SucroseSalts

(Harpers)VitaminmixCholineVitamin

Bj2Control6.0

25.061.75.0

2.00.35.0

MgAmount

(gmjLow

lipotropc6.025.0

62.05.0

2.00.00.0

Mg

Composition of the diets used.

RESULTS

Tables 2 and 3 list results of administration of 100 mg/kg oflasiocarpine to rats on Day 13 of gestation. Five of eightfemales fed the control diet and dosed with the alkaloid onDay 13 succumbed, three of them on Day 17 and one each onDays 18 and 19. Some of these females were examined shortlyafter death, and most fetuses had died prior to the demise ofthe mother. Of the three females surviving, one was killed onDay 20 with all fetuses alive, two were killed on Day 22 withone entire litter dead, and the other with both live and deadfetuses present in the uterus.

Eight females were fed the low lipotrope diet and dosedwith lasiocarpine. One of the eight had no evidence ofpregnancy, four of the eight succumbed on consecutive Days17-20, two had resorbed the conceptus, and two had deadfetuses in utero. The animal killed on Day 20 had resorbed,one of the two killed on Day 22 had both live and deadfetuses, and the other had resorbed.

Lasiocarpine administered to mother rats fed the control orlow lipotrope diet caused maternal liver necrosis which wasmore severe in the animals fed the low lipotrope diet (Figs. 1,2). Some lasiocarpine-treated females had medial hypertrophyof the pulmonary arteries (Fig. 3) irrespective of dietaryregimen, and large, bizarre cells were observed in the lungs ofmany of them (Fig. 4). These changes appeared to have been abit more numerous in the low lipotrope-treated rats, but noeffort was made to quantitate them. Fetal and placentalweights were decreased on both diets as a result of theexposure to lasiocarpine (Table 3). The total number offetuses was reduced in the low lipotrope-treated groups. Twoof twenty-four were dead in control-treated, while 3/10 weredead in the low lipotrope-treated groups, the formerrepresenting two litters and the latter representing a singlelitter. There was a higher incidence of liver necrosis in the lowlipotrope groups (5/13) compared to control-treated fetuses(1/24). Acute liver necrosis and hemorrhage werecharacteristic lesions observed in most fetuses in which lesionswere observed, but some exhibited only bland, parenchymalcell degeneration. Lipid accumulation was usually present inthe liver of fetuses born by dams fed the low lipotrope diet(Figs. 5-8).

Tables 4 and 5 list results from the series in which femalerats were fed the control and low lipotrope diets, dosed with75 mg/kg body weight, and killed on Day 21. Three of eightfemales fed the control diet and dosed with lasiocarpine

aborted, 1/8 died on Day 20, and 4/8 had both live and deadfetuses (Tables 4â€”5)when killed on Day 21. Three of eightfemales fed the low lipotrope diet and dosed with lasiocarpinedied, two on Day 20 and one on Day 21. The fetuses of allthree of these had died prior to the demise of the mother. Oneof eight aborted, one was not pregnant, and 3/8 had both liveand dead fetuses when killed on Day 21.

Lasiocarpine resulted in smaller fetuses (Table 5) with moresevere reduction in weight in those whose dams were fed thelow lipotrope diet. Placenta weight was decreased aboutequally in both groups. Characteristic necrosis and hemorrhagewere observed in the livers of all control-treated females, in6/8 of low lipotrope-treated females, in 21/41 fetuses whosemothers were fed the control diet and dosed with lasiocarpine,and in 19/33 fetuses exposed to the low lipotrope diet andlasiocarpine. Females of both groups had the previously described large cells in the lungs. In general, the effects of thislevel of treatment were more marked than those observed atthe higher level of 100 mg/kg body weight, perhaps a result ofthe better survival of mothers and fetuses in this group whichpermitted a more profound response to treatment. The furtherenhancing effect of the low lipotrope diet was also more pronounced in this experiment.

A third series of pregnant rats was exposed to lasiocarpine,70 mg/kg body weight, in divided doses of 35 mg/kg each ongesta tional Days 13 and 17. One control and twocontrol-treated females were killed on Day 20 to observe theeffects of this level of lasiocarpine on near-term fetuses (Tables6, 7). Neither of the treated females had liver necrosis (Table6), but the fetal livers exhibited characteristic lasiocarpinedamage (Figs. 9, 10). One control-treated and two lowlipotrope-treated females died two to four days postpartum,and the remainder were carried on experiment until the youngwere weaned. The treated-control female, dying four dayspostpartum, had liver necrosis, and 1/5 killed 30 dayspostpartum had liver damage, but the remaining four hadmorphologically normal livers except for an occasionalenlarged liver cell nucleus. One low lipotrope-treated female,killed on Day 20 had liver necrosis, as did both of thosesuccumbing at two and four days postpartum. Liver necrosiswas observed in some of the offspring from these litters.However, one low lipotrope-treated female had no livernecrosis while her offspring did (Figs. 11, 12). Compared tothe two higher dose experiments, both the females and thefetuses on this experiment were considerably less affected bylasiocarpine regardless of the dietary treatment. In contrast tothe two prior experiments, most females in the thirdexperiment gained weight during pregnancy. In the two priorexperiments, most treated females had a net loss of weightfollowing exposure to lasiocarpine. There were fewer dead orstillborn fetuses and the fetal and placental weights weredecreased only in the low lipotrope-treated group. Fetal livernecrosis did not differ significantly between the two groupswith 3/19 and 3/20 on control and lipotrope diets respectivelyhaving microscopic evidence of liver damage. It is significantthat in some of the females a dose schedule on two gestationaldays resulted in no detectable maternal liver damage over andabove that induced by diet, but fetal liver deterioration wasinitiated in utero.

2328 CANCER RESEARCH VOL. 28



Diet and Lasiocarpine Effects

Table 2

FemaleDiet ratNo.Control

556557558559560561562563564565566567Low

lipotrope 568569570571572

573574575576577578579Lasiocarpine(mg/kg)00001001001001001001001001000

000100100100100100100100100Wt.

change(gm)+91+87+39+101-45-22+10+21-28+15-3+10+129

+95+97+98-45

-21-27-40-17-20+19+10Day

ofdemise2020222218a2017a19a2217a2217a20

20222220

2020a19a222218a17aFat000002+002+02+02+

2+2+2+0

1+03+1+01+1+LiverNecrosis00001+2+4+2+2+2+1+1+00001+1+1+4+4+1+2+1+OtherLive

fetusesLivefetusesLivefetusesLivefetusesDeadfetusesLivefetusesDeadfetusesDeadfetusesDeadfetusesDeadfetusesLive

andresorbedDeadfetusesLive

fetusesLivefetusesLivefetusesLivefetusesNot

pregnantFetusesdeadResorbedResorbedResorbedLive

and deadfetusesDeadfetusesDead

fetuses

Effect of low lipotrope diet and lasiocarpine on pregnant rats treated on Day 13."Day on which animal died.

Table 3

Averagewt.Diet

LasiocarpineMaternalControlControl

+Low

lipotropeLowlipotrope+FetalControl+Low

lipotropeLowlipotrope +(gm)+80-6+105-19Live19242410Mortality0/45/80/44/8FetusesDead0203LiverFat

Necrosis0/4

0/44/43/88/83/85/84/4

0/44/45/88/8 1/87/8Average

Average wt.Placenta(gm)wt.(gm)3.7

0.592.80.433.20.522.6

0.35LitterLiveLiveLive

anddeadLiveLive

and deadResorbed ordeadLiverNecrosis0/191/240/245/13

Effect of low lipotrope diet and lasiocarpine, 100 mg/kg body weight on Day 13 of gestation (died prior to or were

killed on Day 20 or Day 22).

Surviving offspring from this experiment have been kept ontheir respective diets for long-term studies and will be reportedat a later date. A few from each group were killed seven weeksafter birth and some of these had interesting liver lesions. Inthe control dietary groups exposed in utero, there was bileduct proliferation and enlargement of parenchymal cell nuclei

in the periportal zone (Fig. 13) and enlargement of nuclei inthe centrilobular zone (Fig. 14). Similar but more severechanges were seen in the periportal area of the lowlipotrope-treated animals (Fig. 15). Toward the centrilobularzone, megalocytes and hyperplasia of the parenchymal cellswere observed (Fig. 16).

NOVEMBER 1968 2329



P. M. Newberne

Table 4

DietControlLow

lipotropeFemalerat

No.671672673674675676677678679680681682683

684685UOD687688689690691692693694Effect

of low lipotrope diet and lasiLasiocarpine(mg/kg)0000757575757575757500007575757575757575Wt.

change Dayor{gm)demise+113+137+117+119-18-8-22-2-14+20+19+29+112

+107+109+105-1+9+3-15-3+8+10+721212121212120a212121212121212121212121212120Â°21Â«20"LiverFat00002+2+01+1+2+1+1+3+4+3+3+2+1+1+01+000Necrosis

Other0

Livefetuses0Livefetuses0Live and deadfetuses0Livefetuses2+

Aborted2+Aborted1+

Deadfetuses2+Live and deadfetuses1+

Aborted1+Live and deadfetuses1+Live and deadfetuses1+Live and deadfetuses0Live fetuses

0 Livefetuses0Live and deadfetuses0

Livefetuses2+Live and deadfetuses0Live and deadfetuses1+Live and deadfetuses0Notpregnant1+

Aborted1+Deadfetuses1+Deadfetuses1+Deadfetusesiocarpine

on pregnant rats treated on Day14."Dead on this gestationalday.DietMaternalControlControlLow

lipotropeLow

lipotropeFetalControlControlLow

lipotropeLowlipotropeLasiocarpine_+-+_+-+Table

5Average

wt.change(gm)

Mortali+

1200/4+11/8+

1080/4+2

3/8FetusesLive

Dead20

033821218

15tyLiverFat

Necrosis0/4

0/47/88/84/4

0/44/8

6/8Average

wt.(gm)5.32.55.01.8Litter4/4

Live3/8Aborted4/8

Live anddead3/4Live

1/4 Live anddead3/8Dead1/8

Notpregnant1/8Aborted3/8

Live anddeadAverage

placentaLiverwt.(gm)necrosis0.58

0/200.3821/410.570/230.36

19/33

Effect of low lipotrope diet and lasiocarpine, 75 mg/kg body weight on Day 14 of gestation (killed on Day 21).

DISCUSSION

Many plants have a well-known poisonous effect on the liverand would not be included in the human diet under ordinarycircumstances. Cirrhosis and liver cancer have been prevalent

in many areas of the world for a long time and speculationsabout their etiology have covered a broad range of factorsincluding infectious diseases, parasites, and malnutrition. Inrecent years, however, there has been some doubt that thesefactors alone could account for the incidence of the diseases so





Table 6

Diet RatNo.Control

755756757758759760761762763764765766Low

lipotrope 767768769770771772773774775776777778Lasiocarpine

(mg/kg)0000707070707070707000007070707070707070Wt.change(gm)+123+130+142+122+93+18+53+45+53+33+64+8+130+135+64+138+63+64+79+35+59+50+24+34Fat0000001+000003+1+2+1+3+3+2+3+2+3+2+2+LiverNecrosis0000000001+02+000003+0002+01+OtherKilled

at Day20Killed1 month,postpartumKilled1 month,postpartumKilled1 month,postpartumKilledat Day20Killedat Day20Killed1 month,postpartumKilled1 month,postpartumKilled1 month,postpartumKilled1 month,postpartumKilled1 month,postpartumDied

4 dayspostpartumKilled

at Day20Killed1 month,postpartumKilled1 month,postpartumKilled1 month,postpartumKilledat Day20Killedat Day20Killed1 month,postpartumKilled1 month,postpartumKilled1 month,postpartumDied

2 dayspostpartumKilled1 month,postpartumDied

4 days postpartum

Effect of low lipotrope diet and 2 doses of lasiocarpine, 35 mg/kg each, on gestational Days 13 and 17.

prevalent in many countries. Until recently, little attention hasbeen paid to toxins of plant or fungal origin, alone or incombination with dietary inadequacies, as etiologic agents inhuman disease. The surge of interest in mycotoxins occasionedby the discovery that mold toxins can represent a seriouspotential hazard to man and animals (19) resulted fromoutbreaks of disease in animals. These epizootics haveawakened plant and animal scientists to the need for researchin this important area of toxicology.

The realization that the Senecio alkaloids, in addition totheir practical importance as an economic problem inlivestock, may be causative factors of some human liverdiseases has resulted in renewed interest in this group ofhepatotoxins. Some of the alkaloids may be suitable for thestudy of the mechanism of the evolution of tumors inexperimental animal models. However, before such studies canbe conducted, it must be confirmed in more than onelaboratory and with several strains of rats that the alkaloids areindeed carcinogenic. The profound delayed impact of theaction of lasiocarpine and other members of the group on theliver indicates the need for an energetic attack on the problemof the mechanism of liver damage whether or not the toxinsare carcinogenic. Since the initial action may take place in theearly stages of life in the rat, it seems likely that infantile liverdisease might result from contamination of human or cow's

milk. This hypothesis is supported by the interestingobservation of Schoental (13) that the administration oflasiocarpine to lactating rats, in doses having no apparenteffects on the mothers, caused fatal liver lesions in the young.We have now shown that in utero exposure to lasiocarpine cancause fetal liver damage without appreciable maternal liverinjury.

Spatz and Laqueur (18) have induced tumors in rats bytransplacental exposure to crude cycad material, andCarnaghan (5) has shown that a single dose of aflatoxin to ratsearly in life can cause liver cancer that appears two years ormore after exposure to the carcinogen. Experiments withpregnant rats indicate that aflatoxin does not result in liverdamage to the rat fetus (our own laboratory, unpublished dataibut this question needs further investigation since Ellis andDiPaolo (7) have demonstrated a positive teratogenic effectin hamster embryos. The teratogenic effects of heliotrine reported by Greene and Christie (8) have been referred topreviously.

Dietary factors cannot be ignored in the study ofcarcinogenesis any more than viral infections and parasites.The induction of nutritional cirrhosis potentiates the liver ofrats to the carcinogenic action of aflatoxin (11) and diets lowin lipotropic factors result in a shorter induction time and ahigher incidence of tumors associated with aflatoxin (12).Population groups, particularly in technologically developingcountries, often consume diets which are low in protein andlimiting in sulfur-containing amino acids. Evidence isaccumulating to show that in many areas of the world thosewho are subject to dietary deficiencies are also exposed todietary contaminants as well as hepatotropic viral infectionsand parasites; these factors must be considered in epidemiologie surveys as well as in basic research aimed at theelucidation of the nature and causes of liver disease and cancer(10,12,19).

The results of studies reported in this presentation indicatethat the maternal diet can appreciably alter the effect of atoxin on the developing embryo and can result in anenhancement of liver changes several weeks after birth. This

NOVEMBER 1968 2331



P. M. Newberne

Table 7

DietLasiocarpineMaternalControlControl

+Low

lipotropeLow lipotrope+FetalControlControl

+Lowlipotrope

Low lipotrope +Average

wt.change(gm)

Mortality+129

0/4+621/8+116

0/4+502/8FetusesLive

Dead27

029241

024 6LiverFat0/41/84/4

8/8Average

wt.(gm)3.73.63.9

2.5Necrosis

Litter0/4

4/4Live2/87/8Live1/8

Live anddead0/44/4 Live

3/8 5/8 Live3/8 Live anddeadAverage

placenta Liverwt. (gm)necrosis0.54

0/270.563/19Â«0.65

0/410.48 3.20"

Effect of low lipotrope diet and lasiocarpine, 2 X 35 mg/kg, Days 13 and 17 of gestation."Some kept on experiment.

observation parallels, in part, the findings of Schoental andMagee (16) in which a diet low in protein accelerated the toxiceffect of a pyrrolizidine alkaloid administered to rats.

The pyrrolizidine alkaloids have been shown to havepowerful mutagenic action in the fruit fly Drosophilamelanogaster (6), and Greene and Christie (8) have inducedmalformations in rats by dosing the mother during variousstages of pregnancy. The natural occurrence of these mutagensin plants presents many questions relative to their continuousaction after ingestion. Those not fatally affected by thehepatotoxic action might sustain genetic deterioration whichcould manifest itself at a much later time when prior exposurewould be unknown or unsuspected.

Consideration of the hepatotoxic effects of lasiocarpine andthe other Senecio alkaloids, as well as their carcinogenicpotential and the enhancing effects of dietary modifications,makes these substances important tools in the study of liverdisease and possibly in cancer. It appears extremely importantto confirm the carcinogenic potential of the major alkaloids,to determine the effects of cofactors such as dietary treatmentand other known liver toxins or carcinogens on potentiation ofthe action of the alkaloids, and to better define similar anddissimilar biochemical and morphologic effects of toxins andcarcinogens. Such studies are in progress in our laboratories.

REFERENCES

1. Bras, G. Berry, D. M., and Gyorgy, P. Plants as Aetiological Factorin Veno-occlusive Disease of the Liver. Lancet, 1: 960-962, 1957.

2. Bull, L. B. The HistolÃ³gica!Evidence of Liver Damage from Pyrrolizidine Alkaloids. Australian Vet. J., 31: 33-40, 1955.

3. Bull, L. B., Dick, A. R., Keast, J. C., and Edgar, G. An Experimental Investigation of the Hepatotoxic and Other Effects on Sheep ofConsumption of Heliotropium europaeum L: Heliotrope Poisoningof Sheep. Australian J. Agri. Res., 7: 281, 1956.

4. Campbell, J. G. An Investigation of the Hepatotoxic Effects in theFowl of Ragwort (Senecio jacobea, Linn.) with Special Referencesto the Induction of Liver Tumours with Seneciphylline. Proc. Roy.Soc. Edinbu., 66: 111-130, 1957.

5. Carnaghan, R. B. A. Hepatic Tumours and Other Chronic LiverChanges in Rats Following a Single Oral Administration of Afla-toxin. Brit. J. Cancer, 21: 811-814, 1967.

6. Clark, A. M. Mutagenic Activity of the Alkaloid Heliotrine inDrosophila. Nature, 183: 731-732, 1959.

7. Ellis, J., and DiPaolo, J. A. Aflatoxin Bj : Induction of Malformations. Arch. Pathol., 83: 53-57, 1967.

8. Greene, C. R., and Christie, G. S. Malformations in Fetal RatsInduced by the pyrrolizidine Alkaloid Heliotrine. Brit. J. Exptl.Pathol., 42: 369-378, 1961.

9. Hill, L. R. Discussion on Seneciosis in Man and Animals: TheWorld-wide Distribution of Seneciosis in Man and Animals. Proc.Roy. Soc. Med., 53: 281-283, 1960.

10. Newberne, P. M. Carcinogenicity of Aflatoxin-contaminated Peanut Meal, In: G. N. Wogan (ed.), Mycotoxins in Foodstuffs, pp.187-208. Cambridge: M.I.T. Press, 1965.

11. Newberne, P. M., Harrington, D. D., and Wogan, G. N. Effects ofCirrhosis and Other Liver Insults on Induction of Liver Tumors byAflatoxin in Rats. Lab. Invest., 15: 962-969, 1966.

12. Newberne, P. M., Rogers, A. E., and Wogan, G. N. HepatorenalLesions in Rats Fed a Low Lipotrope Diet and Exposed to Aflatoxin. J. Nutr., 94: 331-343, 1968.

13. Schoental, R. Liver Lesions in Young Rats Suckled by MothersTreated with the Pyrrolizidine (Senecio) Alkaloids, Lasiocarpineand Retrosine. J. Pathol. Bacterial., 77: 485-495, 1959.

14. Schoental, R. Aflatoxins. Ann. Rev. Pharmacol., 7: 343-356,1967.

15. Schoental, R., and Head, M. A. Progression of Liver Lesions Produced in Rats by Temporary Treatment with Pyrrolizidine(Senecio) Alkaloids and the effects of Betaine and High CaseinDiet. Brit. J. Cancer, 11: 535-544, 1957.

16. Schoental, R., and Magee, P. M. Chronic Liver Changes in Ratsafter a Single Dose of Lasiocarpine, a Pyrrolizidine (Senecio) Alkaloid. J. Pathol. Bacterial., 74: 305-319, 1957.

17. Selzer, G., and Parker, R. G. F. Senecio Poisoning Exhibiting asChiari's Syndrome, A Report on Twelve Cases. Am. J. Pathol., 27:

885-907,1951.18. Spatz, M., and Laqueur, G. L. Transplacental Induction of Tumors

in Sprague-Dawley Rats with Crude Cycad Material. J. Nati. CancerInst., 38: 233-245, 1967.

19. Wogan, G. N. Chemical Nature and Biological Effects of the Aflatoxins. Bacteriol. Rev., 30: 460-470, 1966.





Fig. 1. Photograph of liver section from a female fed the control diet, dosed with lasiocarpine 100 mg/kg body weight on Day 13, and killed onDay 20. Widespread necrosis and hemorrhage were present in all lobes of the liver. H & E, < 140.

Fig. 2. Photograph of liver section from a female fed the low lipotrope diet, dosed with lasiocarpine 100 mg/kg body weight on Day 13, andkilled on Day 20. Characteristic hemorrhagic necrosis was present in all lobes of liver with even more widespread distribution than that observed indosed, control females. Lipid accumulation can be observed in lower left portion of photograph. H & E, x 140.

Fig. 3. Pulmonary artery from a female fed the control diet and dosed with lasiocarpine. Medial hypertrophy was observed in some animals fromeach group irrespective of dietary treatment but venoocclusive lesions of the liver were not observed in any of the animals. H & E, x 400.

Fig. 4. Characteristic large, bizarre cells (arrows) seen in lungs of a number of the females dosed with lasiocarpine. Dietary treatment did notappear to be related. H & E, x 400.

Fig. 5. Liver section from fetus taken on Day 21 from a female fed the control diet. Parenchyma! cells are normal for this stage of development.H & E, x 400.

Fig. 6. Liver section of fetus taken on Day 21 from a female fed the control diet and dosed with lasiocarpine 75 mg/kg body weight on Day 14;lesion primarily necrosis. H & E, x 140.

Fig. 7. Higher magnification of an area from Fig. 6 illustrating parenchyma! cell necrosis. Kupffer cells are still visible, and many sinusoids areintact at this point with lesions in situ. Later, breakdown occurred resulting in large confluent areas of necrosis. H & E, x 400.

Fig. 8. Liver section of fetus taken on Day 21 from female fed low lipotrope diet and dosed with 75 mg/kg body weight on Day 14. Lipiddroplets and severe hemorrhagic necrosis are present. Compare to Fig. 7. H & E, x 400.

Fig. 9. Photograph of a liver section from a female fed the control diet and dosed with lasiocarpine, 35 mg/kg body weight on Days 13 and 17 ofgestation and killed on Day 20. Liver necrosis is not present, but a small amount of hydropic change is evident. H & E, x 400.

Fig. 10. Photograph of liver section taken from the fetus born to the female referred to in Fig. 9. Parenchyma! necrosis is similar to thatobserved in offspring from control-treated females with maternal liver lesions. H & E, x 400.

Fig. 11. Photograph of liver section from a female fed the low lipotrope diet and treated otherwise as the female referred to in Fig. 9. Thecharacteristic large droplet lipid accumulation is the only morphologic evidence of injury H & E, x 400.

Fig. 12. Section of liver from the fetus born to the female of Fig. 11. Although dam had no evidence of liver injury from lasiocarpine, fetal livernecrosis and hemorrhage was widespread. H & E, x 400.

Fig. 13. Periportal bile duct proliferation, parenchyma! cell necrosis, and nuclear enlargement in seven-week-old offspring from a female fed thecontrol diet and dosed with lasiocarpine, 35 mg/kg body weight on Days 13 and 17 of gestation. H & E, x 400.

Fig. 14. Centrilobular area from liver of rat in Fig. 13. There is evidence of slight parenchymal damage and nuclear enlargement. H & E, x 400.Fig. 15. Liver section from seven-week-old rat littered to a female fed the low lipotrope diet and dosed with lasiocarpine 35 mg/kg body weight

on Days 13 and 17 of gestation. Parenchymal cell necrosis, bile duct proliferation, and megalocytosis is present in periportal zone. H & E, x 400.Fig. 16. Centrilobular zone of liver of rat in Fig. 15. Slight lipid accumulation with megalocytosis and early focal regeneration of the

parenchyma are primary features of this area of the liver. Such areas were commonly observed in some of the rats killed at the seven-weekpostpartum period. H & E, x 400.

NOVEMBER 1968 2333



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1968;28:2327-2337. Cancer Res P. M. Newberne and Fetal Rats to LasiocarpineThe Influence of a Low Lipotrope Diet on Response of Maternal

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