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Tunicamycin administered as a single subcutaneous dose of 200 micrograms/kg caused permanent destruction of seminiferous tubules in adult male rats. The fertility of females was unimpaired by doses of up to 450 micrograms/kg. Degenerative changes in seminiferous tubule epithelium commenced 3 to 5 days after injection and by day 19 affected 95% of tubule profiles in sections. In 95% of tubules only Sertoli cells survived to day 56. Tubules without any surviving cells were present from day 19 and slowly increased in number, a variable proportion becoming mineralised. No regeneration occurred within one year. Leydig cells became more prominent because of the atrophy of seminiferous tubules, but their total mass did not differ significantly from that of control rats, nor did the plasma concentration of testosterone. Changes in tissues other than the tests were transient. The testicular damage seems to have resulted from localised ischaemia caused by tunicamycin-induced vasoconstriction.

When dehydroheliotridine (DHH), a pyrrolizidine alkaloid metabolite with bifunctional alkylating and antimitotic activities, was administered to a hooded strain of rats by ip injection, the incidence of tumors, excluding interstitial cell tumors, was significantly greater than that in saline-injected controls. The number of tumors was not further increased when thioacetamide (TA) was co-administered for its mitosis-stimulating effect. The life-span of the rats was significantly shortened by DHH and more so by combined DHH and TA treatment, but not by TA alone. The results indicate that DHH is responsible for some, possibly most, of the carcinogenicity of the parent pyrrolizidine alkaloids and also stimulates the earlier and more rapid development of renal and vascular diseases normally associated with aging in rats.

Local application of dehydromonocrotaline to the rat cremaster produces a delayed prolonged increase in vascular permeability with a time course similar to that of the pulmonary oedema seen after intravenous injection of the same substance. Study of the injured area by the carbon labelling technique and by electron microscopy shows that the increased permeability involves both capillaries and venules of all sizes within the region exposed to dehydromonocrotaline. The vascular leakage appears to be due to a direct effect on the endothelium of small blood vessels. Carbon deposition in labelled capillaries and venules is predominantly intramural and indicative of increased vascular permeability. Accumulation of carbon within the lumen of capillaries is uncommon, and accounts for only a small fraction of total capillary labelling. The findings indicate that capillaries are a major source of inflammatory exudate in this type of injury and suggest that the importance of leakage from capillaries has been underestimated in other types of delayed prolonged increase in vascular permeability.

Female hooded rats were injected intraperitoneally on the 14th day of pregnancy with dehydroheliotridine (DHH), a pyrrolic metabolite of pyrrolizidine alkaloids, at 30 to 90 mg/kg, or with the alkaloid heliotrine at 200 mg/kg and the effects on the embryos were evaluated on the 20th day. DHH was both growth-retarding and teratogenic. A dose of 40 mg DHH/kg approximated 200 mg heliotrine/kg in its effects on embryos, the principal abnormalities being skeletal, including retarded ossification, distorted ribs and long bones, cleft palate and feet defects. At higher dose rates growth almost ceased in many tissues and although more than 60 per cent. of the embryos were alive they would not have survived birth. All were very immature and had multiple skeletal and visceral defects. The effects observed in the liver were anomalous in that the embryonic parenchyma showed little response to the anti-mitotic activity of DHH.

The biological activities of corynetoxins, the causative agents of annual ryegrass toxicity, were compared with those of the closely related tunicamycins and found to be essentially identical. Both showed similar antibiotic activity against Newcastle disease virus and a range of gram-positive bacteria. In preparations of rat liver rough microsomes they also strongly inhibited the uridine diphospho-N-acetylglucosamine (UDP-GlcNAc):dolichol-P N-acetylglucosamine-1-phosphate (GlcNAc-1-P) transferase, an enzyme essential for N-glycosylation of glycoproteins. Pretreatment of rats with corynetoxins resulted in dose- and time-related reduction in the level of activity of this transferase in liver microsomal preparations. The implications of this reduction are discussed with reference to annual ryegrass toxicity, the only field disease known to be caused by tunicamycin-related compounds. Both corynetoxin and tunicamycin produced similar neurological effects and increased vascular permeability in nursling rats and they showed similar LD50-values of 137 and 132 micrograms/kg, respectively, in the nursling rats.

1 Hyperplastic liver nodules were induced in F-344 rats by concurrent administration of lasiocarpine (50 ppm in diet) and thioacetamide (50 mg/kg body weight twice weekly) for 15 weeks. 2 The effect of carbohydrate calorie and total calorie restriction on the fate of hyperplastic liver nodules was examined. 3 The incidence of hepatocellular carcinoma was the same in all groups of rats irrespective of the magnitude of carbohydrate calorie restriction and 50% total calorie restriction. 4 These studies demonstrate that carbohydrate or total calorie restriction has no effect on the progression of hyperplastic nodules to hepatocellular carcinoma.

Eight pyrrolizidine alkaloids of hepatotoxic type have been indentified in leaves of Symphytum X uplandicum. The combined alkaloids exhibit chronic hepatotoxicity in rats.

Pure phomopsin was administered to young Merino x Border Leicester wethers by single subcutaneous (SC) and by single and multiple intraruminal (IR) injection. The toxicity after IR injection was influenced by the size of individual doses and the time over which the total dose was given. At high levels of ingestion the toxicity of phomopsin may be limited by absorption rates; with low daily doses the capacity to repair liver damage may be sufficient to prevent cumulative effects. By SC injection a single dose of 10 micrograms/kg approximated the LD50. By IR injection the overall clinical, biochemical and histological responses closest to these of this SC dose resulted from a single dose of 1,000 micrograms/kg. The same total dose administered at daily rates of 50 or 200 micrograms/kg was more toxic and killed all sheep. A single dose of 500 micrograms/kg caused significant liver damage, but no deaths. Single doses of 125 and 250 micrograms/kg and repeated daily doses of 12.5 micrograms/kg over 16 weeks caused no detectable tissue damage. Inappetence was the most sensitive indicator of phomopsin toxicity. About 10% of the sheep differed substantially from the rest of the flock in their susceptibility to phomopsin poisoning.

The antimitotic action of the pyrrolizidine alkaloid lasiocarpine on rat liver parenchyma was investigated using as the experimental model the wave of mitosis produced in liver by a single dose of thioacetamide. A single low dose of lasiocarpine administered two weeks before the thioacetamide, almost completely inhibited the mitotic wave without inhibiting to the same extent the preceding wave of DNA synthesis. By the use of selective inhibitors and radioisotope labelling, the location of the mitotic block was found to be either in the latter half of the DNA synthetic phase, S, or early in G2, the post-synthetic phase. The mitotic wave was similarly inhibited by pretreatment of the rats with a single injection of dehydroheliotridine, a pyrrolic metabolite of heliotridine-based pyrrolizidine alkaloids.

Study of cleared, histological and electron-microscope specimens shows that increased vascular permeability plays a major role in the formation of the pulmonary oedema and pleural effusions that occur in rats following the intravenous injection of a large dose of dehydromonocrotaline. There is a latent interval of 6-8 hr between injection of the dehydroalkaloid and the start of increased permeability which appears to be due to a direct damaging effect of the toxin on the endothelium of pulmonary capillaries and small venules. The endothelial injury does not cause permanent disruption of small blood vessels, and 2 days after injury all vessels are patent and lined by a complete layer of endothelium. Large numbers of mononuclear cells are present in the interstitial tissues of the lung 44 hr after injury. These cells appear to be emigrated blood monocytes but the cause of their emigration and their role in the subsequent progression of this type of injury to the lung are not clear.

Pyrrolizidine alkaloids have long been considered to be hepatotoxic in man as well as in grazing animals. To investigate the effect of liver cell division induced by thioacetamide on the hepatic changes induced by these alkaloids, rats were treated concurrently with thioacetamide and the pyrrolizidine alkaloid lasiocarpine. Thioacetamide was given intraperitoneally in a dose of 50 mg/kg b. wt twice weekly and lasiocarpine was administered in the diet at a concentration of 50 ppm. At 15 weeks, the combination of thioacetamide and lasiocarpine produced numerous grossly visible grey nodules in livers of 26 of 30 rats. Microscopically, these livers revealed a severe degree of postnecrotic cirrhosis and numerous hyperplastic nodules. The cells in most nodules were arranged in solid sheets or in a trabecular pattern and shown atypia, mitosis and hyperchromasia. In contrast, there was no evidence of cirrhosis or nodule formation in livers of animals treated with either lasiocarpine or thioacetamide alone. The rapid development of liver lesions in rats treated simultaneously with low doses of lasiocarpine and thioacetamide suggests that cell proliferation accentuates the development of neoplasia.ł

62 pyrrolizidine alkaloids and derivatives have been screened for acute and chronic hepato- and pneumotoxicity by the single dose method previously described. This procedure is satisfactory for the compounds of medium to high hepatotoxicity but failed to detect toxicity in certain other compounds of known, low hepatotoxicity. New findings significant in relation to hepatotoxicity are as follows: (i) On a molar basis, diesters of heliotridine and retronecine are about 4 times as toxic as the respective mono-esters and heliotridine esters are 2-4 times as toxic as retronecine esters. (ii) Crotanecine esters are less toxic than retronecine esters, and the 6,9-diester madurensine, 2-4 times less toxic than the 7,9-diester anacrotine (the difference being ascribed to there being only one reactive alkylating centre in the toxic metabolite from madurensine). (iii) Hepatotoxicity was confirmed for 7-angelylheliotridine but not observed for 9-angelyheliotridine and 7- and 9-angelylretronecine. (iv) Other significant compounds failing to induce hepatotoxicity were 9-pivalyl- and 7,9-dipivalyheliotridine, the alpha- and beta-epoxides of monocrotaline, 7-angelyl-1-methylenepyrrolizidine and the methiodides of monocrotaline and senecionine. The following compounds are readily converted by rat liver microsomes in vitro into dehydroheliotridine (or dehydroretronecine): 7- and 9-angelyheliotridine, 7- and 9-angelylretronecine, 7,9-dipivalylheliotridine and otosenine. 7,9-Divalerylheliotridine, the alpha- and beta-epoxides of monocrotaline, and retusamine yield pyrrolic metabolites more slowly. The preparation and characterisation of several alkaloid derivatives are described. Chronic lung lesions were produced by most compounds which gave chronic liver lesions, although a higher dose was required in some instances. This requirement may sometimes mean that chronic lung lesions cannot be induced because of the intervention of acute or peracute deaths. Apart from this factor, structure activity requirements for pneumotoxicity are the same as for hepatotoxicity, consistent with their being both caused by the same toxic metabolites.

The neurological signs induced by injection of tunicamycin are, in young adult rats, virtually identical to those typical of acute experimental autoimmune encephalomyelitis (EAE). Vasogenic exudation, of which the occurrence in the spinal cord of EAE rats has been shown to coincide with the onset of clinical signs, was investigated by quantitative electroimmunoblotting of central nervous system (CNS) tissue at various times following tunicamycin injection of young adult rats. Highly elevated levels of extravasated plasma proteins were observed in the spinal cord from 48 h after injection and, as in EAE rats, these increases coincided with the onset of neurological impairment. At 72 h post-injection, significant increases were also found in the brain of affected animals, albeit at much reduced levels. This is in contrast to previously reported findings in nursling rats where oedema was shown to be predominantly located in the brain. Quantitative electroimmunoblotting for myelin basic protein (MBP) in the CNS of tunicamycin-treated young adult rats indicated that, as in acute EAE, no extensive demyelination had occurred. These data provided further evidence that in both neurological diseases, vasogenic oedema of the spinal cord may be causally related to the appearance of neurological signs and suggested that its differential localization in the CNS may lead to differential neurological impairment.

The influences of in vivo pretreatment with phenobarbitone (PB), 3-methylcholanthrene (3-MC), 2,2',4,4',5,5'-hexachlorobiphenyl (HCBP), and 3,3',4,4'-tetrachlorobiphenyl (TCBP) on cytocidal hepatotoxicity of two pyrrolizidine alkaloids, lasiocarpine (LC) and senecionine (SC), were compared in short-term primary cultures of rat hepatocytes. Toxicity was measured by release of lactate dehydrogenase (LDH) into culture medium at 24 h. LC was slightly more toxic to control hepatocytes than SC in the graded response range of 10-160 microM. PB and HCBP (a PB-type polychlorobiphenyl inducer) similarly potentiated toxicity of SC, and each diminished the degree to which cell killing by LC and SC was inhibited by SKF-525-A. By comparison, 3-MC and TCBP (a 3-MC-type PCB inducer) each diminished toxicity of SC but had little effect on toxicity of LC. Alpha-naphthoflavone (ANF) potentiated toxicity of both LC and SC in hepatocytes induced by 3-MC or TCBP but had little effect on responses of hepatocytes induced by either PB or HDBP. These results indicate that xenobiotics that induce similar patterns of cytochrome P-450 isozymes have qualitatively similar modulating influences on cytocidal hepatotoxicity of pyrrolizidine alkaloids in primary cultures. However, the observed modulating effects could not be explained solely on the basis of altered activation rates by the cytochrome P-450 species known to be induced by the various xenobiotics.

Extracts of annual ryegrass (Lolium rigidum) infected with Anguina sp. and Corynebacterium sp., and associated with an outbreak of annual ryegrass toxicity in sheep, were administered to 2-week-old rats by a single intraperitoneal injection. Rats that received a lethal dose of toxin developed neurological signs including incoordination and convulsions from the second day and most died between 2 and 7 days after injection. Histologically, the brains showed widespread lesions of focal necrosis consistent with anoxia. Peripheral circulation rate was greatly reduced after 3 days and gangrene of the tail and hind legs developed in some rats. Evidence of restricted blood flow was also seen in kidneys, lungs and brain. It was concluded that the toxin contains a long-acting vasoconstrictor.

Quantitative toxicity studies were carried out in sheep using corynetoxin, tunicamycin and toxic annual ryegrass (Lolium rigidum). Sheep were very sensitive to these toxins. The lethal dose was about 35 micrograms/kg bodyweight for pure tunicamycin given by subcutaneous injection and 3 to 5 mg/kg for corynetoxin administered orally as slurries of bacterial galls of known corynetoxin content. The total lethal dose was of the same order, whether given as a single dose or as repeated smaller doses, the maximum interval tested being 9 weeks between doses. This finding indicates that a second exposure of animals to toxic rye grass in the one season would present a greater risk than would a first exposure to the same field. It also demonstrates the advisability of the monitoring of pasture levels of gall contamination, as levels below those that produce clinical signs of the disease may still contribute to an accumulating burden of toxicity.

Dehydroheliotridine, a major pyrrolic metabolite of heliotridine-based pyrrolizidine alkaloids, interacted with both native and heat-denatured DNA in vitro under mildly acid conditions to form a soluble non-diffusible complex as shown by spectral data. The complex formed with heat-denatured DNA was more resistant than heat-denatured DNA alone to the combined action of deoxyribonuclease and phosphodiesterase. Analysis of the resistant fractions suggested the presence of at least one base not obtained from DNA alone.