Studies on the Toxi ~ ~ ology of the Palmyrah palm ( Borassus flabellifer L ) : Part 11 . Milk ' Transfer of Toxicity to Suckling Rats

Flour from t!le young shoot of the Palmvrah palm is consu.ned by humans in so~ne Aslan anc! African cou.tlries ; their traditional pharmac~poie~ irclude? palmyrah prod.ucts as thera?e:!tic agents. This flour has previovsly becn rqortec! to prod.uce acute and chronic toxic effects in ~ats . We regort herc thst feedin2 of diets containing palmyrah flour to lactating rats resalted in hepatic, pa!mona.ry a d renal lesions with subcvtaneo~rs haemonhages in the suckling ,-ats. The lesions were mainly varcular \ ~ . i t l ~ diffuse haemorrhages or severe sinusoida! congestion in the liver, 11a.emorrhages in the pulmo~ary alveolar septa with non-expansion of thc l:tngi anc! intertubular congestion in the kidneys. With 100 %, 50 % and 33 % flour diets, lethnlity occurred in the neonates within one week, 10 days and two weeks respentively. The significznce of these results on liver damage in hnman infants through maternal consumption of this flour is discussed. 1. htl-oduction Flour from the yol~ng shoot of the palmyrah palm (Rorassus,flabeIl~f~v L) is consuined by h~1.rnan.s in some tropical co~lntries including Sri Lanka, both a food and as a trad.itiona.1 medicine. Ti is flour wl~.en fed to rats, was reported to produce hepatotoxic and neurotoxic effects with letha!ity after short term, high d.ose feedingi central an.d portal veno-occlusive reactions with hepatic fibrosis5 and imnm~tnosuppressive effects2 on prolonged. feed.ing. Toxic compounds, in.cluding a few of pla.nt origin, have been rep~r ted . '>~>~ to be excreted through milk and. it was therefore of interest to investigate a possible transfer of palmyrah toxicity tlzrougl~ =ilk as a possible route of intoxication. o f young animals which are also reported to be more susceptible to some hepatotoxic agents. This report describes the investigation of the effects of the consumptioll of palmytah flour by lactating rats, on their suckling offspring; we report that intoxication resulting in hepatic, pulmonary and renal lesions wit11 lethality were produced in the suckling neonates. 278 S. AT. Avseczllerutne, A . A. L. Gunatilaka and R. G. Pnnnbolcke


htl-oduction
Flour from the yol~ng shoot of the palmyrah palm (Rorassus,flabeIl~f~v L) is consuined by h~1.rnan.s in some tropical co~lntries including Sri Lanka, both a food and as a trad.itiona.1 medicine.Tiis flour wl~.en fed to rats, was reported to produce hepa- totoxic and neurotoxic effects with letha!ity after short term, high d.ose feedingi central an.d portal veno-occlusive reactions with hepatic fibrosis5 and imnm~tnosuppressive effects2 on prolonged.feed.ing.
Toxic compounds, in.cluding a few of pla.nt origin, have been rep~rted.'>~>~to be excreted through milk and. it was therefore of interest to investigate a possible transfer of palmyrah toxicity tlzrougl~ =ilk as a possible route of intoxication. of young animals which are also reported to be more susceptible to some hepatotoxic agents.
This report describes the investigation of the effects of the consumptioll of palmytah flour by lactating rats, on their suckling offspring; we report that intoxication resulting in hepatic, pulmonary and renal lesions wit11 lethality were produced in the suckling neonates.

Experimental
2.1 Flour.Flour was obtalned by pulverising, recently processed (boiled and sundried) mould-free shoots which were in the form in which they are normally consumed by humans in this country.The flour was stored at 4OC to prevent microbial spoilage.Flour from 5 different batches of shoots was tested.No contamination with aflatoxin or commonly used agrochemicals (organophosphates, cl~lorinated hydrocerbons and paraquat) was detected in these batches.
Palmyrah flour has a calorie equivalent of 340.6 Kca1/100 g, producing a calorie intake of 50 Kcal/l5O g rat/day nit11 a 100 % flour dietary consumption of 15 g per day.
2.2 Rats and feeding.Pregnant Wistar rats were ma-intained in individual cages and fed. on their normal diet of rat pellets (approximate consumption -15 g/rat/day) with water cld lib., till parturition.Immediately after parturition, the test diet (15 glratlday) of I00 %, 50 % or 33 % palmyrah flour in pellet powder was introduced with water ad lib.Th.e experiments were terminated well before weaning began and this together with th.e inaccessibility of the food to the young, excluded the possibility of direct ingestion of the test diet by the neonates.Normal infants of age, from mothers fed.with similar amounts of pellet powder alone, were used.as controls for histopathology.
Fourteen lactating rats and 86 infants were studied.; histological studies were made on 44 of the infants.

2.3
Histology.Tissues from infant rats (either killed with di-ethyl ether or examined soon after d.eath from intoxication) were fixed in 10% formol-saline for paraffin embedding.Sections 5 , ~ in thickness were stained with haematoxylin and eosin (H & E) and reticulin stains.
The livers of intoxicated lactating rats which died during the course of the experiments or were killed after the d.eath of all the infants, were also examined simiIarIy for histological confirmation of palmyrah toxicity.

3.1
General effects.The toxicity of the flour was confirmed by the development of the typical symptoms of palmyrah toxication in the mother rats-pilo-erection, excitability, spasms of the limbs and head., followed by inactivity, laboured respiration and death and by histology of the liver which showed centrilobular sinusoidal congestion.These effects were most marked with 100 % and 50 %flour within 5-10 days of feeding.Intoxicated infants did not show the neurotoxic symptoms (excitability and spasms) even when the mother was on a 100% flour diet, but appeared less active had a shrivelIed skin and retardation of growth.
Palmjirah toxins in n?il?c 3.2 Lethality.With 100 % flour in the maternal d~et, neonatal deaths occurred within the first and seventh day.With 50 % and 33 :d diets, survival was prolonged to approximately 10 days and 2 weeks respectively.The survival period thus appeared to be dose dependent.None of the infants of the palmyrah fed mothers, at any of the 3 dose levels survived beyond the second week.
3.3 External abnormalities.In 8 infants which survived for more than 4 days with a maternal diet of 100 % flour, subcutaneous haemorrhages appeared 2t the extremities of the tail and.limbs, and in the thoracic and abdominal walls.Spontaneous nmputation of the discoloured tail or limb extremities occurred in some of these infants.
3.4 Internal abnormalities.In fatal cases, the neonatalliver was darker than in normal infants of the same age; the discolouration was unevenly distributed among the liver lobes.Free pleural or peritoneal fluid was seen in 3 infants; the kidneys were enlarged and pale in 2. Neither intestinal !laemorrhage nor oedema of the viscera, which were described by Schoenta16 in pyrrolizidine alkaloidal intoxicztion, was seen in the palmyrah intoxicated infants.These abnormalities were seen with 100% and 50% maternal palmyrah diets.
3.5 Histopathology.In general, the intensity of the histopathological abnormalities in the infants appea.red to be proportional to the concentration of the flour in the maternal diet, rather than to the duration of survival.
The discoloured patches in the tail, thoracic and abdominal walls and in the limbs consisted of extensive subcutaneaus haemorrhages although no clear evid.ence of vascular disruption to account for the haemorrhages, was detected.. In the tail, the haemorrhage was accompanied by oedema of the subcutaneous tissues and, necrosis of the cartilaginous cells.
3.6 Liver.The most marked and consistent neonatal histological abnormalities were seen in the liver.Mild to intense centrilobular sinusoidal congestion with gross dilation of the sinusoids and focal h.aemorrhages, witb 100 % and 50 % flour, were the most obvious lesions (Figure 1).Except with 100% flour when the vascular abnormalities were intense and diffuse throughout the Iiver lobes, lower doses of flour produced an irregular distribution of these lesions, some lobes appesring relatively unaffected.This correlated with the macroscopic discoloration of the liver lobes in these mfants.The focal nature of the haemorrl~ages contrasted with extensive centrilobular pools of extravasated blood which are reported to be seen in nitrosamine intoxicated adult rats.
Neither central venous disruption, veno-occlusive reactions whch are seen in adult rats fed on sub-lethal doses of palmyrall flour, nor thromboses of the vein.s,were seen in our infant rats.With extensive congestion and l~aernorrhages: extra-medullary erytllropoetic foci in the liver were absent.

3.7
Lllrmps.Vascular congestion was the commonest lesion.With 100% and.50% flour, the congestion was severe, accompanied by scattered foci of haemorrhage into the alveolar septa; in 8 rats which showed these lesions, the apices of the lungs showed macroscope congestioli or l~aemorrhage.Non-espansion of the alveoli which was present in 10 neon.atesappeared to correlate with the d.egree of vascular congesl.ionand haem.orrl~age and could have resulted, from a depression of respiration with intoxication.
In S infant? the intensity of the congestion and haemorrhage into the liver was not paralleled by the degree of congestion in the lungs, suggesting that the liver lesions were a direct toxic effect on the vasculature rather than a passive effect from central venous congestion.

Spleen.
In only 1 infant d.id the spleen show vascu1a.rcongestion.The absence of splenic congestion in infant rats which sho:vzd hepatic vascular abnormalities, also supports the possibility that the hepatic lesions were a direct toxic effect of the palnlyrah flour.
3.9 I<idney.Mild to moderate intert~tb~llar vascular congestion was the only renal lesion in 13 infants.

Discussion
Schoenta16 in-vestigated. the effect on suckling rats of pyrrolizidine alkaloids (PA) administered.parenterally to lactating motl~ef rats and reported.that with the doses used, the liver lesions in the infants were most marked at 3 to 7 weeks of 2ge.bToteworllzy differences between her findings and ours inclnded the occurrence of intestinal haemorrhages, fatty changes in the liver with bile duct proliferation cln.c! central venous lesions (thickening of the venous walls wit11 narrowing of the 1urn.en) in PA poisoning.We have de~cribed.~differences between the palinyrah ind:xed lesions in ad.ull rats and those of PA poisonin%; these together with our findings described in this report further support our coilclusion that palmyrah toxicity is not due to pyrrolizidine alkaloids.
A further difference between Schoental's PA lesions and. the palmyrall ind.uced lesions was that with tile doses used, PA did not p r o d ~~c e intoxication in the 111otl3.e~rat although lesions were well marked in the suclljng infants.In our experiments.the lactating mothers too showed simultaneons clinical and histopathological.evidence of palmyrall intoxication.The infant rats in our experiments showed nlacroscopic and microscopic evidence of intoxication within the first week of life contrasting with, the delay of 2 to 3 weeks in the development of PA lesion observed by Schoental.Whether this difference is due to a d.ose related effect or to differences betbeen PA and palmyrah toxin [s) in their modes of action, is at pesent obscure.
We have observed (Arseculeratne, unpl~blished d.zta ) that death with.1.00% flour ocsurred more rapidly with younger rats [I to 2 months of age ) than in adult rats; this might suggest that infant rats i. 11 th? present experinzents d.eveioped more inten.selesions than th.eir mothers on account of aged.ependentsusceptibility, as has been described of other hegatotosins, notably zflatoxin and the pyrrolizidi~le alkaloids.
The histologici~l lesions too, in the live1 and the lungs of our infant rats were Inore intense than the lesions in the corresponding mother rats.Whether this indicates a. greater susceptibility of the infants, the excretion tll.roug!l milk of a more toxic metabolite, a concentlation of the original palmyral:.toxin ir! nl!'lk sncll as through bind.ing to millc proteins or an.activation of tile original flour toxin during its passa.gethrough the mamma.rygland, remains to be elucidated.

S. N. Arseculeratlze, A. A. L. Gunatilaka and R. G. Panabokke
We have previcusly remarked the relatively insignificant hepatocellular abnormalities even in fdtal cases of palmyrah intoxication in adult rats, the main lesions having been in the vasculature.In the intoxicated infznt too, the 11epatoceUular lesions were not co-extensive with the vascular lesions.This contiasts \\it11 the effects of nitrosamines and aflatoxin.
Other plant sources, toxins from whch have been shown to be transferred through milk incl~tde locoweeds4, Bracken fern3, and Cycus sp.
Our findings may also provide an alternative approach to the isolation and identification of the palmyrah toxins as excreted though milk, and the investigation of a possible metabolic transformation of flour toxins into more toxic derivatives.
These findings also suggest that maternal consumption of palmyrah flour may result in toxic liver damage in infants which may contribute to the lelatively high incidence of ~hronic liver disease jn humans in Asian and African countries.