The heart and pulmonary arteries in rats fed on Senecio jacobaea

SHORT ARTICLES

THE HEART AND PULMONARY ARTERIES I N RATS FED ON SENECIO J A C O B A E A

JAMES BURNS Department of Pathology, University of Liverpool

PLATE XC

THE seeds and foliage of Senecio jacobaea, commonly called “ ragwort ”, contain several pyrrolizidine alkaloids, namely seneciphylline, senecionine, jacobine, jaconine, jacoline and jacozine (Bull, Culvenor and Dick, 1968). The pyrrolizidine alkaloid, monocrotaline, has been shown to produce hypertensive pulmonary vascular disease in rats when it is added to their diet (Kay and Heath, 1966; Heath and Kay, 1967; Kay, Harris and Heath, 1967). In view of this, the following investigation was undertaken to see whether powdered Senecio jacobaea was also a cause of right ventricular hypertrophy and hypertensive pulmonary vascular disease.

MATERIALS AND METHODS

Thirty female adult Wistar albino rats, weighing between 80 and 110 g, were divided into three groups, A, B and C, each consisting of ten animals. All were allowed free access to powdered rat cubes and water. In groups A and B the diet was adulterated with powdered dried Senecio jacobaea consisting of the stem, leaves, flowers and seeds of the plant, which had been harvested in September 1969 and allowed to dry naturally in the air for a period of 4 mth. The powder was added to give a concentration of 5 per cent. of the diet in group A, and 10 per cent. in group B. The ten rats in group C were kept as controls. Test rats were allowed to die spontaneously and necropsy was performed as soon after death as possible. The control rats and the surviving test rats were killed on the 57th day of the experiment.

All the animals were weighed at the outset of the experiment, three times per week during its duration, and at death.

At necropsy the thoracic viscera were removed en bloc and fixed in 4 per cent. formalde- hyde. The lungs were distended through the trachea with the same solution until the pleural surfaces were smooth. After fixation the heart was dissected free and opened, and blood and excess fixative were removed by blotting with fine gauze. The heart was dissected according to the method of Fulton, Hutchinson and Jones (1952). The individual weights of the right ventricle, the left ventricle together with the interventricular septum, and the atria together were measured. The total heart weight was obtained by addition of these three values. To assess the significance of the weight of the right ventricle it was convenient to express it as its inverse ratio to the weight of the left ventricle and interventricular septum ((LV+S)/RV); this ratio had the great advantage that it was independent of total heart weight and, in the absence of left ventricular hypertrophy, provided a reliable assessment of the presence or absence of right ventricular hypertrophy (Fulton et al.).

The pulmonary trunk and aorta were dissected free from the heart and embedded in paraffin wax. Transverse sections of both vessels were cut at 5 pm thickness and stained by Lawson’s modification of the Weigert-Sheridan method for elastic fibres and with a Van Gieson’s mixture as counterstain for collagen and muscle. In each case the medial thickness

Received 11 Mar. 1971; accepted 19 July 1971. I. PATH.-VOL. 106 (1972) 187

188 JAMES BURNS

of the vessel was measured. Ten measurements of the medial thickness were made on each vessel and from these the mean medial thickness was calculated. The mean medial thickness of the pulmonary trunk was expressed as a multiple or submultiple of the mean medial thickness of the aorta (FT/A ratio), which gave a reliable indication of the presence or absence of any increase in the thickness of the tunica media of the pulmonary trunk.

Paraffin sections, 5 pm thick, from the left lung and from each of the superior, middle, inferior and median lobes of the right lung were stained with Ehrlich's haematoxylin and aqueous eosin, and by Lawson's modification of the Weigert-Sheridan method for elastic tissue with a Van Gieson's mixture as counterstain, and by the prussian blue method for ferric iron. Measurements of the medial thickness of the " muscular pulmonary arteries " were made in all animals. These arteries consist of an intima of endothelial cells and a thin uniform media of circularly orientated smooth-muscle fibres sandwiched between internal and external elastic laminae. They possess a fibrous adventitial coat and their external

"01

Body w t (g)

IJ Controls - A 5 per cent. ragwort in diet X X l l

10 per cent. ragwort in diet X X I X 50

L

pl

I 1

0 10 20 30 40 50 60 0 1 ,

Survival time (days)

FIG. 1.-Growth curves of test rats compared with those of controls.

diameter ranges from 20 to 350 pm (Kay and Heath, 1969). Whenever possible ten such vessels were measured in each lobe, but only arteries that were virtually circular in transverse section were measured. The diameter was taken as the mean of two measurements, at right-angles to each other, of the distance between diametrically opposite points on the external elastic lamina. The medial thickness was estimated as the mean of four measurements taken at approximately equally spaced points around the vessel wall, and expressed as a percentage of the external diameter of the vessel. A value for the average percentage medial thickness in each animal was obtained by totalling all the mean percentage medial thicknesses and dividing the sum by the number of vessels examined.

A qualitative assessment was made of the pulmonary arterioles in the test and control animals. These vessels are arterial branches that have a muscular media at their origin but none in their remaining portion. Their diameter is less than 20 pm and they arise as ter- minations or side branches of muscular pulmonary arteries. Their walls consist of endothelial cells and a single elastic lamina (Kay and Heath, 1969).

BLIRNS PLATE XC

PULMONARY HYPERTENSION IN RATS FED ON RAGWORT

FIG. 2.-Control rat. Muscular pulmonary artery. The media is thin. Elastic and Van Gieson (EVG). x480.

FIG. 3. -Test rat. Muscular pulmonary FIG. 4.-Test rat. P~tlnionary arteriole. Develop- artery. Thickening of the media. EVG. ment of a muscular media and an external elastic x 480. lamina. EVG. x 1500.

PULMONARY HYPERTENSION IN RATS FED ON RAGWORT 189

RESULTS

Macroscopic appearances The rats whose diet contained Senecio jacobaea failed to gain weight normally (fig. 1).

They became weak, and four of them (B5-7, B10) died between the 40th and 50th days of the experiment. At necropsy their lungs presented a red-and-white mottled appearance. One animal (B10) had serous pleural effusions. No abnormalities were noted in any of the control animals.

TABLE I Evidence of right ventricular hypertrophy in rats fed on a diet

containing dried ragwort (Senecio jacobaea)

Diet

Containing 5 per cent. dried Senecio jacobaea


Without added Senecio jacobaea

Rat no.

A1 A2 A3 A4 A5 A6 A7 A8 A9 A10

B1 B2 B3 B4 B5 B6 B7 B8 B9 B10

c 1 c 2 c 3 c 4 c 5 C6 c 7 C8 c 9 c10

Cardiac weights (mg)

Total

301 289 345 470 265 323 467 279 41 5 337

289 245 294 298 453 392 482 28 1 315 470

43 1 473 428 405 479 430 420 45 1 449 478

LV+S*

23 1 220 210 233 204 226 268 228 243 246

196 177 218 167 277 214 278 198 160 242

299 330 319 299 366 323 342 366 365 345

64 55

117 220 46 82

175 49

150 70

74 56 62

128 141 158 131 148 135 172

97 87 88 85 68 68 53 66 (34 80

LV+S RV -

3.6 4.0 1.8 1 .o 4.4 2.7 1.5 4.6 1.6 3.5

2.6 3.1 3.5 1.3 1.9 1.3 2.1 1.3 1 *5 1.4

3.1 3.8 3.6 3.5 5.4 4.6 6.4 5.5 5.7 4.3

* LV+S = Weight of left ventricle and interventricular septum. t RV = Weight of right ventricle.

Histological appearances Qualitative data

Muscular pulmonary arteries. In the control rats the " muscular pulmonary arteries " With three exceptions (A2, B3, B4) the " muscular pulmonary were thin-walled (fig. 2).

190 JAMES BURNS

arteries ” of the test rats showed an obvious increase in medial thickness even before con- firmation by measurement (fig. 3).

Pulmonary arterioles. No abnormality was noted in any of the control animals. In all the test animals some of the pulmonary arterioles were thick-walled, owing to the develop- ment of a layer of circularly orientated smooth muscle lying between the normal elastic lamina and a newly formed external elastic lamina (fig. 4).

TABLE I1 Evidence of medial pulmonary hypertrophy in rats fed on a diet

containing dried ragwort (Senecio jacobaea)

Diet



Without added Senecio jaco baea

Rat no.

__ ~-

A1 A2 A3 A4 A5 A6 A7 A8 A9 A10

B1 B2 B3 B4 B5 B6 B7 B8 €39 B10

c 1 c 2 c 3 c 4 c5 C6 c 7 C8 c 9 c10

Mean medial thickness (pm) of

PT* _ _ _ _ .

82.8 114.4 102.1 140.8 65.1 59.0

115.9 81.6

129.5 91-3

~- ~

108.5 115.5 67-1 67.3

106.4 165.4 108.2 131.8 86.5

143.9

40-5 39.4 45.8 35.4 40.7 35.6 37.4 41.8 44.0 36.1

136.0 182.8 106.7 1 19.7 123-6 103.6 105.4 125.4 125.2 104.7

~~

135.0 136.8 103.0 100.8 93.7

136.2 129.1 102.1 1 14.2 112.1

118.8 110.7 122.1 97.5

126.3 I 1 2.6 120.3 127.6 101.0 124.1

PT Ratio -~ A

0.60 0.60 0.90 1.20 0.60 0.50 1.10 0.60 1 .oo 0.80

0.80 0.80 0.60 0.60 1.15 1.20 0.80 1.20 0.70 1.28

0-34 0.35 0.37 0.36 0.32 0.32 0.3 1 0.33 0.43 0.29

* PT = Average medial thickness of pulmonary trunk. 7 A = Average medial thickness of aorta.

Quantitative data Weight of right ventricle. In the control animals values ranged from 53 (C7) to 97 mg (Cl)

(table I). In the test animals values ranged from 46 (A5) to 220 mg (A4). In the control animals the ratio of the weight of the left ventricle and interventricular septum to the weight


of the right ventricle ranged from 3.1 (Cl) to 6.4 (C7) (table I); in the test animals it ranged from 1.0 (A4) to 4.6 (A8).

Medial thickness ofpulmonary trunk. In the control animals the mean medial thickness of the pulmonary trunk ranged from 35.4 (C4) to 45.8 pm (C3) (table 11); in the test animals the range was from 59.0 (A6) to 165.4 pm (B6).

The FT/A ratio ranged from 0.29 (C10) to 0.43 (C9) in the control animals and from 050 (A6) to 1.28 (B10) in the test animals (table 11). Figure 5 shows the relation between the PT/A ratio and the mass of the right ventricle. Ingestion of Senecio jacobaea is associated with a concomitant increase in the medial thickness of the pulmonary trunk and right ventricular hypertrophy.

" O l

A A A 0 A

PT

A 0.75 -

A 1, 0.

0.50 I I 0.25

= 5 per cent. ragwort in diet

A:= 10 per cent. ragwort in diet

o= Control

0 0 0

0 0 1 2 3 4 5 4 7 8

(LV + S)JRV

FIG. 5.-Relation between the FT/A ratio and the inverse ratio of the weight of the right ventricle,

Medial thickness of muscular pulmonary arteries. The range of the external diameter of the " muscular pulmonary arteries " examined in the control rats was between 22 and 121 pm and the range of their average medial thickness was 3.3 (C5) to 5.4 (Cl) per cent. of the external diameter (table 111). Between 37 and 50 " muscular pulmonary arteries " were examined in each of the test animals. The range of the external diameter of the vessels examined was between 22 and 165 pm and the range of their average medial thickness was 4.9 (A2) to 16.3 (B5) per cent. of the external diameter (table 111).

Figure 6 shows the relation between the thickness of the media of the " muscular pulmonary arteries" and the mass of the right ventricle. Ingestion of Senecio jacobaea is associated with concomitant increase in medial thickness of muscular pulmonary arteries and right ventricular hypertrophy.

DISCUSSION All but seven of the test rats (Al, A2, A5, A8, A10, B2 and B3) showed all three morbid

anatomical criteria of pulmonary arterial hypertension, viz., right ventricular hypertrophy and medial hypertrophy of the pulmonary trunk and muscuiar pulmonary arteries. Even these seven exceptions, however, showed increased medial thickness of the pulmonary trunk. Furthermore, all but two (A2, B3) showed medial hypertrophy of the muscular pulmonary

192 JAMES BURNS

arteries. Hence, all the test rats showed some morbid anatomical evidence of pulmonary hypertension, although in seven instances this had not led to right ventricular hypertrophy, presumably because the pulmonary arterial hypertension was of insufficient duration. These results indicate that Senecio jacobaea contains an agent that producespulmonary hypertension.

TABLE I11 Further evidence of increase in medial thickness of muscular pulmonary arteries

in rats fed on a diet containing dried ragwort (Senecio jacobaea)

Diet


Containing 10 per cent. dried Senecio jncobaea

Without added Senecio jacobaea

Rat no.

Al A2 A3 A4 A5 A6 A7 A8 A9 AIO

B1 B2 8 3 B4 B5 B6 B7 B8 B9 B10

c 1 c2 c3 c4 C5 C6 c7 C8 c 9 c10

Muscular pulmonary arteries

Number examined

38 38 50 50 44 37 50 45 50 41

38 45 50 50 50 50 50 50 50 50

50 50 50 50 50 50 50 50 50 50

Xange of external diameter (pin)

30.0-1 10.0 27.5-165.0 24.2-1 37.5 24.2- 99.0 22.0- 77.0 26.4-1 21 -0

24.2- 67.1 264-126.5

25.3- 92.4 23.1- 59-4

24.2- 70'4 23-1- 96.8 24'2-1 29.8 23.1- 81.4 30'0-146'0 264- 80-3 25.3- 88.0 24.2- 88.0 23.1-110'0 30'8-107.8

23.1-1 10.0 24.2- 90.2 26.4- 66.0

24.2- 99.0 23.1-113.3

24'2-103.4 25.3- 86.9 24.2-121'0

22.0- 69,3

22.0- 86.9

Mean per cent. medial thickness

12.1 4.9

10.5 9.6

14.2 10.5 11.2 10.6 13.9 12.8

10.9 11.7 5.5 6.1

16.3 14.1 11.4 10.8 7.2

16.0

5.4 4.0 4.4 4.6 3.3 3.4 3.9 3.8 3.7 4.4

In view of the fact that monocrotaline, a pyrrolizidine alkaloid, has been shown to produce this haemodynamic effect (Kay et al., 1967) it is likely that the agent in Senecio jacobaea is one or more of the pyrrolizidine alkaloids contained in the plant.

Although the toxic effects of plants belonging to the genus Senecio have been well- recognised for many years (Willmot and Robertson, 1920; Cook, Duffy and Schoental, 1950; Selzer and Parker, 1951; Harding et al., 1964; Lidberg, 1966; Schoental, 1968) the production of hypertensive pulmonary vascular disease by one of these plants or their alkaloids has not been described before. The demonstration that monocrotaline (Kay et al., 1967) and fulvine (Kay et al., 1971a) produce hypertensive pulmonary vascular disease


in rats when added to their diet is consistent with a concept of dietary pulmonary hypertension.

This effect of the pyrrolizidine alkaloids on the pulmonary circulation is important because it raises the possibility of a dietary cause for at least some cases of pulmonary hypertension in man. In this context there has been great interest in cardiological circles in Europe, over the past few years, in the possible association between the introduction as an anorexigen of aminorex fumarate, which is not a pyrrolizidine alkaloid, and the subsequent increase in incidence of primary pulmonary hypertension in the centres where it has been used. Though there is strong statistical evidence that aminorex fumarate causes hypertensive pulmonary vascular disease (Gurtner, 1969; Rivier, 1970; Greiser and Gahl, 1971) animal experiments have so far failed to confirm this (Kay, Smith and Heath, 1971b).

20 = 5 per cent. ragwort in diet

15

MT (per cent.) 1C

I

A 0

A == 10 per cent. ragwort In diet

0 = Control 0

0 0

A A

O A

I 1 2 3 4 5 6 7 8

(LV + S)/ RV

FIG. 6.-Relation between the thickness of the media of the muscular pulmonary arteries and the inverse ratio of the weight of the right ventricle.

Whatever the eventual outcome of this controversy, the fact that some pyrrolizidine alkaloids can produce hypertensive pulmonary vascular disease and right ventricular hypertrophy in rats suggests that a careful dietary history should be taken when dealing with cases of primary pulmonary hypertension in clinical practice. A disturbing feature that came to light during the course of the present study was the fact that I was able to purchase a large quantity of dried chopped Senecio jucobueu from a " Health Stores " in the Liverpool area. The material purchased was meant to be taken in the form of an infusion to cure a variety of ailments.

SUMMARY All of a group of twenty rats fed on a diet containing 5 or 10 per cent. of powdered

Senecio jucobaea (ragwort) showed anatomical and histological evidence of hypertensive pulmonary vascular disease.

I wish to thank Professor D. Heath and Dr J. M. Kay for suggesting this study and for their helpful advice and constructive criticism during its course. In addition, I wish to

194 JAMES BURNS

thank Dr V. Walters and Mr J. F. Humphreys-Jones of the Department of Pharmacy, Liverpool Polytechnic, for providing the facilities and technical assistance for powdering the Senecio jacobaea.

REFERENCES BULL, L. B., CULVENOR, C. C. J., AND DICK, A. T. 1968. The pyrrolizidine alkaloids. Their

chemistry, pathogenicity and other biological properties, Amsterdam. COOK, J. W., DUFFY, E., AND SCHOENTAL, REGINA 1950. Primary liver tumours in rats

following feeding with alkaloids of Senecio jacobaea. Br. J. Cancer, 4,405. FULTON, R. M., HUTCHINSON, E. C., AND JONES, A. M. 1952. Ventricular weight in cardiac

hypertrophy. Br. Heart J., 14, 413. GREISER, E., AND GAHL, K. 1971. Frequency estimation of ingestion of aminorex and

incidence of primary pulmonary hypertension in a defined population. Proc. XI1 Ann. Meet. Eur. SOC. Study Drug Toxicity, Uppsala, Sweden, June, 1970, Amsterdam, Excerpta med., Znt. Congr. Ser., no. 220.

GURTNER, H. P. 1969. Pulmonary hypertension produced by ingestion of substances. Editorial, Bull. Physio-Pathologie respiratoire, 5 , 1969.

HARDING, J. D. J., LEWIS, G., DONE, J. T., AND ALLCROFT, R. 1964. Experimental poisoning by Senecio jacobaea in pigs. Path. vet., 1, 204.

HEATH, D., AND KAY, J. M. 1967. Medial thickness of pulmonary trunk in rats with cor pulmonale induced by ingestion of Crotalaria spectabilis seeds. Cardiovasc. Res., 1, 74.

KAY, J. M., HARRIS, P., AND HEATH, D. 1967. Pulmonary hypertension produced in rats by ingestion of Crotalaria spectabilis seeds. Thorax, 22, 176.

KAY, J. M., AND HEATH, D. 1966. Observations on the pulmonary arteries and heart weight of rats fed on Crotalaria spectabilis seeds. J. Path. Bact., 92, 385.

KAY, J. M., AND HEATH, D. 1969. Crotalaria spectabilis, the pulmonary hypertension plant, Springfield, Ill.

KAY, J. M., HEATH, D., S m , P., BRAS, G., AND SUMMERELL, JOAN 1971a. Fulvine and the pulmonary circulation. Thorax, 26,249.

KAY, J. M., SMITH, P., AND HEATH, D. 1971b. Aminorex and the pulmonary circulation. Thorax, 26,262.

LIDBERG, L. 1966. An experimental study of veno-occlusive disease. Acta SOC. Med. upsal., 71,263.

RMER, J.-L. 1970. Hypertension arterielle pulmonaire primitive. Schweiz. med. Wschr., 100, 143.

SCHOENTAL, REGINA 1968. Toxicology and carcinogenic action of pyrrolizidine alkaloids. Cancer Res., 28,2237.

SELZER, G., AND PARKER, R. G. F. 1951. Senecio poisoning exhibiting as Chiari’s syndrome: report on 12 cases. Amer. J. Path., 27, 885.

WILLMOT, F. C., AND ROBERTSON, G. W. 1920. Senecio disease or cirrhosis of the liver due to Senecio poisoning. Lancet, 2, 848.

The heart and pulmonary arteries in rats fed on Senecio jacobaea

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