Part - 1 : Studies with myoporaceous essential oils.
(1). Single doses of the myoporaceous essential oil ngaione in rats cause hepatocellular necrosis which has an unusual midzonal pattern. Previous studies in this laboratory in which ngaione was dosed daily to male rats beginning with a sublethal dose which was increased weekly by 50% showed that the animals eventually became tolerant to daily doses of the oil in excess of the LD50, and with no liver necrosis occurring. In the present study, this dosing regime for ngaione was used in male mice. The animals became similarly tolerant to daily LD50 doses of the oil. Within the first week of dosing there were a few small foci of necrosis in the midzonal parenchyma but this did not increase with subsequent increase in the daily dose level, and the hepatic parenchyma had became generally resistant to the necrogenic action of the oil. Susceptibility to ngaione dosing at the LD50 level returned at from 4 to 7 days after cessation of daily ngaione dosing, at which stage the zone of injury was now periacinar rather than midzonal in location.
(2). Earlier studies of ngaione dosing in the mouse and rat had shown that pretreatment of the animals with sodium phenobarbitone caused a translocation of the zone of liver necrosis from midzonal to periportal. In addition when ngaione and phenobarbitone were administered daily to male rats, with the same kind of dosing regime for ngaione as above, the animals again became tolerant to the necrogenic doses of the oil until daily doses in excess of the LD50 were reached. Within 1 to 2 weeks of treatment the hepatocytes had arranged themselves int© distinctly periacinar and periportal populations. The latter then assumed the appearance of regeneration nodules. Eventually with increasing oil dose, the periacinar parenchyma gradually became damaged and disappeared, to be eventually replaced by regeneration nodules derived from the newly proliferated periportal parenchyma. This gave the liver the appearance of cirrhosis.
This same procedure was applied to male mice. The animals became increasingly resistant to ngaione with increasing dose and again the hepatic parenchyma resolved itself into two zones as for the rat. However, the periportal parenchyma did not develop into regeneration nodules within the 3 weeks period of dosing as occurs in the rat. Return to normal susceptibility to ngaione occurred within 7 days of cessation of dosing but the zone of necrosis was periacinar despite the continued daily exposure to phenobarbitone. Within a few days the periacinar zone again became resistant to ngaione while necrosis was again seen in the periportal zone. It was clear that the use of simultaneous dosing of phenobarbitone and ngaione would not produce a satisfactory model of cirrhosis in this species.
(3). Of the naturally occurring furanosesquiterpenoid essential oils found in Myoporum deserti, only deisopropylngaione (DIN) was able to produce extrahepatic as well as liver lesions, namely lung damage in both sexes and kidney damage in male mice. Animals grazing Myoporum plants occasionally die as a result of severe pulmonary lesions and renal lesions have also been reported. DIN may be the cause of such lesions. An experiment was accordingly carried out in mice in which the animals were dosed daily with DIN, beginning with a sublethal dose, increased by 50% weekly as in repeated dose studies above. Some mice survived for 5 weeks by which time the daily DIN dose was about twice the predose LD50. In these animals there were no lesions found in the liver, lungs or kidneys, indicating that mice could become completely tolerant to daily exposure to lethal doses of DIN. As for the hver in which suicidal destruction of the P45o-mediated toxic action of the furans is considered to occur, lesions in the lungs and kidney were not produced for probably similar reasons. Furan-induced damage in these tissues also requires P450-induced toxic activation.
(4). As for repeated simultaneous dosing with ngaione and phenobarbitone in the mouse, the same kind of study was carried out using DIN and phenobarbitone. Pretreatment of mice with phenobarbitone did not reduce the susceptibility of the animals to a single dose of DIN as occurs with ngaione. However, multiple dosing of sublethal levels of DIN did induce tolerance to the oil as seen in the previous experiment. With the highest dose used of about twice the pretreatment LD50, deaths occurred due to liver necrosis, lung and kidney lesions being slight or not present. Again toxic metabolism of the furan in the extra hepatic tissues appeared to be suppressed following multiple DIN dosing as for the previous study.
(5). In an earlier study in this laboratory a cirrhosis model was developed in the male rat by means of simultaneous dosing with sodium phenobarbitone and ngaione. The animals were dosed with p^nobarbitone for one week and then given daily intraperitoneal doses of ngaione, beginning with a sublethal dose and then with weekly 50% increases until daily doses in excess of the LD50 could be given. A suitable model for cirrhosis had been developed earlier, using simultaneous dosing with phenobarbitone and CCI4, the latter dosed twice weekly. More recently this model had been refined such that the CCI4 dose was given once weekly to each animal depending on the effect of the previous single exposure on body weight change. Cirrhosis developed within 5 weeks. This procedure was applied to the phenobarbitone - ngaione model to see if such an improvement in yield of cirrhotic animals could be obtained with such a short period. Since the lesion caused in the liver by ngaione after phenobarbitone pretreatment was periportal in location this was considered to be superior to a model in which the basic site of injury was periacinar. In addition the ngaione was dosed by the oral route in lieu of the intraperitoneal. The preliminary ngaione dose was determined according to the CCI4 published method and this was given initially to the phenobarbitone pretreated animals.
The ngaione dose selected, dosed once weekly for 5 weeks did not appear to be leading to the degree of injury experienced in the CCI4 model. The ngaione was then dosed to effect in individual rats but tolerance to ngaione in the periportal parenchyma appeared to occur, and it was only at high doses that hepatocellular injury occurred. Necrosis was highly unpredictable in extent and degree as some animals were able to survive for up to 30 LD50 doses of the oil over some 4 months. In these animals the original hepatic parenchyma had been replaced by regeneration nodules but possible consequences of portal hypertension such as ascites did not develop. The livers of various rats sacrificed at different times after cessation of ngaione dosing continued to consist of nodular formations but sinusoids had developed wjthin the solid nodules, providing for increased perfusion of these tissues. The combination of phenobarbitone and ngaione given simultaneously to the young male rat did not yield a cirrhosis model as expected. This was probably mainly due to the ability of the hepatocyte to develop tolerance to this compound following even intermittent exposure, for different reasons, in both the periportal and periacinar parenchyma.
Part - 2 : Studies with Ageratina adenophora.
(1). Regular consumption by horses of Ageratina adenophora leads to chronic lung injury, reduced exercise tolerance and death in up to 2 years. Following unpublished reports that the inclusion of leaves of the plant in the diet of mice leads, not to lung damage but to liver damage, studies were carried out in mice to confirm this finding. Mice fed diets containing 10 to \6% of the powdered leaf developed focal hepatocellular necrosis associated with injury to the small bile ducts. The condition was associated with intense jaundice. A feature of the necrotic foci was that replacement and repair of this tissue was very slow. A comparison was made in mice of the repair of the zonal liver lesion caused by respiratory exposure to CCI4 in which repair was complete within 10-14 days. In the Ageratina dosed mice necrotic hepatocellular foci were still present 50 days after the start of plant feeding.
(2). Following these preliminary studies of the toxic effects of the plant, chemical investigation, based on an assay procedure developed from this work revealed that the hepatotoxic principle in the Ageratina leaves was the cadenine sesquiterpene ketone, 9- oxo-10,ll-dehydroageraphorone. This had an oral LD50 for mice of about 350 mg/kg. Initial studies confirmed the nature of the hepatotoxic effect as a focal hepatocellular necrosis associated with damaged bile duct walls from which bile could readily escape into the regional parenchyma. This lesion is similar to that caused by isothiocyanates in which the biliary tract injury is GSH dependant. When mice livers were depleted of GSH by means of pretreatment with buthionine sulfoximine and diethyl maleate, dosing with ageratina toxin did not cause biliary tract lesions but periacinar hepatocellular necrosis. Pretreatment with sodium phenobarbitone as well as induced* reduction in hepatic GSH resulted in midzonal hepatocellular necrosis. This suggested that ageratina toxin, at least partly, needed metabolism for toxicity to hepatocytes.
The structure of the ageratina toxin was similar to pulegone, the toxic essential oil from pennyroyal. The latter compound undergoes P450-mediated metabolic transformation to menthofuran and this in turn is metabolised to a y-ketoenal which is the toxic electrophile. Ageratina toxin may well also be converted to an analogue of menthoftiran and as a furan be able to cause midzonal hepatic necrosis, as for ngaione. A mechanism is suggested based on this hypothesis and the analogy with the isothiocyanates for the toxicopathology of ageratina toxin.
Part - 3 : Studies with Persea americana.
(1). The toxicity of avocado leaves for lactating goats had been experimentally established and the changes in the mammary gland described. In the present studies, the same leaf material was used to study the effects of the plant in lactating mice. It was found that inclusion of as little as 5% of freeze dried powdered leaf in the diet would result in failure of the litter to gain weight. This was shown to be due to necrosis of the lactating epithelium. Mice which succumbed to this toxicity also had hydrothorax and pulmonary oedema. Histologically, myocardial necrosis also occurred.
(2). This study was followed by an avocado leaf intoxication of two male goats dosed with 9.1 and 6.2 g/kg of freeze dried leaf respectively. One animal died within 48 hours with hydrothorax and pulmonary oedema. Observations made on the remaining animal included thoracic radiography, echocardiography and ECG. This animal also died within 48 hours of dosing and necropsy revealed pleural and pericardial effusion, pulmonary oedema and myocardial injury. Avocado toxicity was clearly associated with cardiotoxicity and this confirmed literature reports of field and experimental studies in other species.
(3). Further studies of the effects of a single 24 hour exposure to 5% avocado leaf in the diet of lactating mice revealed that the severity of the effects on the mammary gland ranged from no apparent effect to complete necrosis of the secretory epithelium and loss through starvation and thirst of the litter within 3 days. There were intermediate degrees of the effect which resulted in transient weight loss of the litter and then recovery of normal growth rate. Histological examination of such mammary glands revealed loss of whole lobules, with involution and replacement with scar tissue. These effects on the litter were mostly without significant effect on the clinical appearance of the dam.
(4). Preliminary attempts were made to isolate the toxic fraction (s) from the avocado leaf, using the procedure of Harbourne (1984) for isolation of alkaloids from plant material. No toxic alkaloids were isolated from the plant and the results of the extractions indicated that the toxin (s) were present in a preliminary petroleum ether extract. The test animals used were male mice, which of course, were unsatisfactory for isolating compounds toxic for the lactating mammary gland. In this assay death from cardiotoxicity was the end point used.
(5). Following the use of an assay model developed from the preliminary work in mice fed avocado leaf supplemented diet, chemical investigations in our laboratory revealed the toxic principle for the lactating mammary gland to be (Z,Z)-1-acetyloxy-2- hydroxy-12,15-heneicosadien-4-one which was given the trivial name persin. This had the oral toxicity of 60 - 100 mg/kg. This compound was then dosed to several lactating mice on the 4th to 8"^ day of lactation and the results recorded. The toxic effects of persin on the lactating acinar epithelium were confirmed and the effects on the glands with varying degrees of severity of toxicity recorded.
Under the influence of suckling on such glands, significant cellular proliferation in affected acini occurred. This hyperplasia was considered to be derived from residual acinar stem cells. These formations were gradually removed during the course of the lactation through apoptosis of the individual cells and at the conclusion of lactation no trace of the hyperplastic nodules remained. These changes in the lactating mammary gland do not appear to have been described previously.