Peter R. Cheeke

Species differences in the hepatic microsomal enzyme metabolism of the pyrrolizidine alkaloids

Species differences in pyrrolic metabolites and senecionine (SN) N-oxide formation among eight animal species (sheep, cattle, gerbils, rabbits, hamsters, Japanese quail, chickens, rats) varying in susceptibility to pyrrolizidine alkaloid (PA) intoxication were measured in vitro by hepatic microsomal incubations. The results suggested that there is not a strong correlation between the production of pyrrolic metabolites and susceptibility of animals to PA toxicity. The rate of PA activation in hamsters, a resistant species, measured by formation of (+/-)6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP) far exceeded the rate of SN N-oxide formation (detoxification) (DHP/N-oxide = 2.29). In contrast, SN N-oxide was the major metabolite in sheep, another resistant species, with much lower production of DHP (DHP/N-oxide = 0.26). The roles of cytochrome P450s and flavin-containing monooxygenases (FMO) in bioactivation and detoxification of pyrrolizidine alkaloids (PA) were studied in vitro using sheep and hamster hepatic microsomes. Chemical and immunochemical inhibition data suggested that the conversion of SN to DHP is catalyzed mainly by cytochrome P450s (68-82%), whereas the formation of SN N-oxide is carried out largely by FMO (55-71%). There also appeared to be a high rate of glutathione-DHP conjugation in hamster (63%) and sheep (79%) liver microsomal incubation mixtures. Therefore, low rates of pyrrole metabolite production coupled with glutathione conjugation in sheep may explain the resistance of sheep to SN, whereas the high rate of GSH-DHP conjugation may be one of the factors contributing to the resistance of hamsters to intoxication by this PA.

Protective effects of butylated hydroxyanisole against the acute toxicity of monocrotaline in mice

Abstract Dietary butylated hydroxyanisole (BHA) at levels of 0.25 and 0.75% was found to protect young female mice against the acute toxicity of monocrotaline. The protective effect was associated with reduced levels of pyrrole metabolites in liver, decreased activity of hepatic aminopyrine demethylase, and a reduced rate of in vitro microsomal conversion of monocrotaline to pyrrole metabolites. BHA also increased liver sulfhydryl levels and the activity of cytosolic glutathione S -transferase. Dietary cysteine (1%) was less protective than BHA against monocrotaline toxicity. The LD50 values of monocrotaline in control and cysteine fed mice were 259 and 335 mg/kg, respectively, but for BHA-fed mice, the LD50 value was 497 (0.25% dietary BHA) or 542 mg/kg (for 0.75% dietary BHA with or without 1% cysteine). Dietary cysteine did not produce an increase in liver sulfhydryls but it increased the glutathione S -transferase activity and the in vitro production of pyrroles from monocrotaline. The ip administration of monocrotaline (280 mg/kg) did not affect liver sulfhydryl levels or aminopyrine demethylase activity but produced a significant decrease in the in vitro formation of pyrroles from monocrotaline. Although alterations in microsomal monooxygenase activities occurred after BHA, cysteine or monocrotaline treatment, the concentration of cytochrome P -450 in liver was unchanged.

Effect of ethoxyquin on the toxicity of the pyrrolizidine alkaloid monocrotaline and on hepatic drug metabolism in mice.

Abstract Diets containing the antioxidant ethoxyquin (6-ethoxy-2,2,4-trimethyl-1,2-dihydroquinoline) were fed to female CD-1 mice for 10–38 days to assess their effects on monocrotaline toxicity, liver glutathione levels and hepatic drug metabolizing enzyme activities. Dietary ethoxyquin (0.25%) protected the mice against lethality as well as acute hepatotoxicity of monocrotaline as measured by the levels of alanine aminotransferase and asparate aminotransferase in plasma. Other feed additives with antioxidant properties such as vitamin C, vitamin E or selenium had no protective effect against monocrotaline lethality and hepatotoxicity. Dietary cysteine (1%) also protected mice against the lethality but not the acute hepatotoxicity of the alkaloid. With the exception of ethoxyquin, none of the other feed additives increased liver glutathione levels (mg/liver). Glutathione S-transferase activity was significantly increased by either dietary ethoxyquin or cysteine using chlorodinitrobenzene as substrate. Dietary ethoxyquin produced an increase in hepatic cytochrome P-450 content and an increase in the in vitro conversion of monocrotaline to pyrrole metabolites by liver microsomes. However, there was no effect of the feed additive on the activity of aminopyrine demethylase and on the concentration of pyrroles found in the liver 2 or 24 h after monocrotaline administration. Since ethoxyquin protected mice against monocrotaline lethality and hepatotoxicity despite no reduction in the in vivo activation of monocrotaline, the mechanisms involved are most probably a result of increased detoxication processes partly because of increased liver glutathione levels.

Consumption of poisonous plants (Senecio jacobaea, Symphytum officinale, Pteridium aquilinum, Hypericum perforatum) by rats: chronic toxicity, mineral metabolism, and hepatic drug-metabolizing enzymes.

Effect of dietary tancy ragwort (Senecio jacobaea), comfrey (Symphytum officinale), bracken (Pteridium aquilinum) and alfalfa (Medicago sativa) on hepatic drug-metabolizing enzymes in rats were measured. Tansy ragwort and bracken increased (P less than 0.05) the activity of glutathione transferase and epoxide hydrolase. Comfrey and alfalfa increased (P less than 0.05) the activity of aminopyrine N-demethylase. Feeding bracken or St. Johns wort (Hypericum perforatum) in conjunction with tansy ragwort did not influence chronic toxicity of tansy ragwort as assessed by rat survival time. Dietary tansy ragwort resulted in increased (P less than 0.05) hepatic copper levels; the other plants did not affect copper levels. The results do not suggest any major interaction in the toxicity of tansy ragwort with bracken or St. Johns wort.

Toxicity of Senecio jacobaea and pyrrolizidine alkaloids in various laboratory animals and avian species

The chronic toxicity of tansy ragwort (Senecio jacobaea) to several herbivorous laboratory animals and to chicks and turkey poults was examined by feeding the dried plant as a component of a mixed diet. Gerbils, hamsters and guinea pigs were resistant to chronic toxicity. Gerbils were highly resistant, consuming over 3500% of their body weight of the dried plant, whereas susceptible species succumb to a tansy ragwort intake of 5-20% of their body weight. Guinea pigs and gerbils were resistant to acute toxicity of injected monocrotaline, a pyrrolizidine alkaloid (PA). They were moderately resistant to acute toxicity of injected tansy ragwort alkaloids. Both chicks and turkey poults were susceptible to chronic toxicity of dietary tansy ragwort.

Effect of pyrrolizidine alkaloids from tansy ragwort (Senecio jacobaea) on hepatic drug-metabolizing enzymes in male rats

Abstract Ad lib. consumption of diets containing 5% tansy ragwort ( Senecio jacobaea ) for 1–4 weeks produced a 5- to 6-fold increase in hepatic microsomal epoxide hydrase and significant increases in cytosolic glutathione S -transferase activities in male Long-Evans rats. An enhancement of these enzyme activities was also observed when a diet containing 1% tansy ragwort was fed for a period of 3 weeks. Feeding a diet containing 0.5% pyrrolizidine alkaloid (PA) mixture extracted from tansy ragwort for 1 week produced a 5-fold increase in hepatic epoxide hydrase and a 73 per cent increase in glutathione S -transferase activities. In contrast, hepatic microsomal aryl hydrocarbon hydroxylase activity (AHH) was reduced significantly by feeding diets containing 5% tansy ragwort or a 0.5% alkaloid mixture. Hepatic microsomal cytochrome P-450 content was lowered following consumption of the 0.5% alkaloid mixture but not by feeding a 5% tansy ragwort diet, the difference presumably being a result of the lowered PA intake by the latter animals. Exposure to the pyrrolizidine alkaloids, therefore, may influence significantly the capacity of animals to metabolize endogenous or foreign compounds and possibly also affect the subsequent biotransformation and toxicity of these plant constituents.

Modifications of chronic hepatotoxicity of pyrrolizidine (Senecio) alkaloids by butylated hydroxyanisole and cysteine

The chronic hepatotoxic effects of mixed pyrrolizidine alkaloids (PAs) from the poisonous plant tansy ragwort (Senecio jacobaea) and the ability of butylated hydroxyanisole (BHA) and cysteine to alter these hepatic effects were studied in male rats. In control animals, the i.p. administration of a single dose of mixed PAs, 160 mg/kg, produced marked fibrosis, biliary hyperplasia, megalocytosis, necrosis and calcification in liver 8 weeks post injection. In contrast, consumption of 0.75% BHA diet 10 days before and 14 days after PA administration reduced the incidence and/or completely prevented the occurrence of these pathological changes. Similar treatment with 1% cysteine, however, only reduced the severity of the hepatic lesions.

The effect of consumption of the pyrrolizidine alkaloid-containing plant Senecio jacobaea on iron and copper metabolism in the rat.

The effect of dietary tansy ragwort (Senecio jacobaea), a pyrrolizidine alkaloid (PA)-containing plant, on mineral metabolism in rats was studied. In experiment 1, rats were fed a dietary level of 5% tansy ragwort. At intervals of 1, 2, 4, 6 and 8 weeks animals were killed and tissue mineral levels determined. As compared to comparable controls, rats fed tansy ragwort showed by 6 weeks elevated liver and spleen copper and iron levels. Experiment 2 was a 3 X 4 factorial experiment with added dietary copper levels of 0, 50 and 250 ppm, and tansy ragwort levels of 0, 1, 2.5 and 5%. Liver copper levels increased markedly with increasing levels of tansy ragwort; at 0, 50 and 250 ppm added copper, liver copper levels were 4, 18 and 21 times greater in rats fed 5% tansy ragwort as compared to those with no tansy ragwort. Increases in liver iron and spleen copper were noted with consumption of tansy ragwort. Higher liver copper levels were observed when a casein-based diet rather than a soybean meal diet was used, suggesting an effect of phytate in soybean meal in reducing copper absorption. In the last experiment, 59Fe was administered to rats fed diets with or without tansy ragwort. After 5 weeks on tansy ragwort, rats showed very low levels of 59Fe in erythrocytes, tibia and liver, and elevated levels in spleen and kidney, suggesting either an impairment of hematopoesis or accelerated erythrocyte destruction as a result of PA consumption. These results indicate that PAs cause increased liver copper content, and disturbances in iron metabolism.

An evaluation of acetone extracts from six plants in the AMES mutagenicity test

Acetone extracts from six plants were evaluated for mutagenic activity with the Salmonella/mammalian-microsome mutagenicity test (Ames) utilizing tester strains TA98 and TA100 and in the presence and absence of induced rat liver microsomes. Extracts from alfalfa (Medicago sativa), lettuce (Lactuca sativa) and thread-leaf groundsel (Senecio longilobus) produced only negative responses. Comfrey (Symphytum officinale) and tansy ragwort (Senecio jacobaea) extracts produced toxic responses that were abolished in the presence of the microsomal bioactivation system. Bracken (Pteridium aquilinum) and tansy ragwort extracts produced positive responses following bioactivation with the liver microsomal system. The results suggest that the Ames mutagenicity test may be of some value in initial evaluations for potential toxic effects of plants consumed by animals and man.

Toxicity of Senecio jacobaea (tansy ragwort) in rats

Abstract To investigate the toxicity of the pyrrolizidine alkaloid containing plant Senecio jacobaea (tansy ragwort), rats were fed diets containing 0 or 5% tansy ragwort and then sacrificed at 1, 2, 4, 6, and 8 weeks. Tansy ragwort produced a depression of body weight gain, food intake, and food efficiency as early as 1 week after beginning treatment. In tansy-fed rats, the relative weights of lungs, heart, spleen, adrenals, and testes were increased at 4 weeks while that of kidney was increased after 6 weeks. Leukocyte counts were markedly elevated at 2 weeks whereas the hematocrit and erythrocyte counts were decreased, respectively, at 4 and 6 weeks of treatment. At 4 weeks, total serum protein, serum albumin, and albumin:globulin ratios were decreased in the tansy-fed rats. The principal pathological changes observed in treated rats were enlarged spleen with lymphoid hyperplasia and increased hematopoietic activity, atrophied thymus, ascites, hydrothorax, and diffuse necrotizing hepatitis. The majority of the gross and histopathological changes were evident following 6 and 8 weeks of treatment.