Barry L. Smith

Mechanism of bracken fern carcinogenesis: Evidence for H-ras activation via initial adenine alkylation by ptaquiloside

Bracken fern (Pteridium spp.) causes cancer of the oesophagus and the urinary bladder in cattle and sheep. Ptaquiloside (PT) is believed to be the carcinogenic principle which alkylates DNA when activated to its unstable dienone form (APT) under alkaline conditions. In this report we present evidence for the presence of PT-DNA adducts in the ileum of bracken fern-fed calves using the 32P-postlabelling assay. H-ras mutations were also observed in the ileum using single strand conformation polymorphism (SSCP) technique. Mutations corresponding to adenine to pyrimidine transversions in the codon 61 of H-ras were identified by the cycle sequencing method. In vitro DNA alkylation studies showed that APT alkylated H-ras primarily at the adenines. In addition, the rate of depurination of alkylated adenine was sequence dependent. Investigation of DNA template activity using a plasmid DNA showed that DNA synthesis by T7 DNA polymerase was terminated by the presence of all alkylated bases but certain apurinic sites allowed the DNA synthesis to continue. These results suggest that initial alkylation of adenine by PT in codon 61 followed by depurination and error in DNA synthesis lead to activation of H-ras proto-oncogene.

EFFECTS OF BUTYLATED HYDROXYANISOLE AND DICOUMAROL ON THE TOXICITY OF MENADIONE TO RATS

The enzyme DT-diaphorase catalyses the 2-electron reduction of quinones. This reaction may facilitate the detoxification of such compounds, since the hydroquinone so formed can be converted into non-toxic conjugates. There is evidence for the involvement of DT-diaphorase in the detoxification of menadione (2-methyl-1,4-naphthoquinone) in a wide range of cells and tissues in vitro, but no information is available on the possible influence of this enzyme on the harmful effects of menadione in vivo. In animals, menadione is selectively toxic to erythrocytes, causing haemolytic anaemia. In the present study, rats were treated with dicoumarol, an inhibitor of DT-diaphorase, or butylated hydroxyanisole (BHA), a substance that increases the activity of this enzyme in vivo. They were then challenged with a toxic dose of menadione. Dicoumarol increased the severity of menadione-induced haemolytic anaemia while BHA decreased it, consistent with a role for DT-diaphorase in the detoxification of menadione in vivo, as previously described in vitro.

COMPARATIVE TOXICITY OF 2-HYDROXY-3-ALKYL-1,4-NAPHTHOQUINONES IN RATS

2-Hydroxy-1,4-naphthoquinone has previously been shown to cause severe haemolytic anaemia and renal tubular necrosis in animals. In order to establish if such toxic effects are common to other 2-hydroxynaphthoquinone derivatives, the short-term toxicity of a number of 2-hydroxy-3-alkyl-1,4-naphthoquinones has been compared in rats. 2-Hydroxy-3-methyl, 2-hydroxy-3-ethyl- and 2-hydroxy-3-propyl-1,4-naphthoquinone were found to cause both haemolysis and renal damage, although the severity of the changes provoked by these substances was much lower than those induced by the parent compound at an equivalent dose-level. Furthermore, the toxicity of the hydroxy-alkylnaphthoquinones decreased with increasing size of the alkyl substituent and no toxic changes were recorded in animals dosed with 2-hydroxy-3-butyl- or 2-hydroxy-3-pentyl-1,4-naphthoquinone. The relationship between the in vivo effects of these substances and previously reported data on their in vitro cytotoxicity is discussed in relation to the mechanism of toxicity of these and other naphthoquinone derivatives.

Effect of inducers of DT-diaphorase on the toxicity of 2-methyl- and 2-hydroxy-1,4-naphthoquinone to rats.

It has previously been shown that rats pre-treated with butylated hydroxyanisole (BHA), a well-known inducer of the enzyme DT-diaphorase, are protected against the toxic effects of 2-methyl-1,4-naphthoquinone but are made more susceptible to the harmful action of 2-hydroxy-1,4-naphthoquinone. In the present experiments, the effects of BHA have been compared with those of other inducers of DT-diaphorase. Rats were dosed with BHA, butylated hydroxytoluene (BHT), ethoxyquin (EQ), dimethyl fumarate (DMF) or disulfiram (DIS) and then challenged with a toxic dose of the naphthoquinones. All the inducers protected against the haemolytic anaemia induced by 2-methyl-1,4-naphthoquinone in rats, with BHA, BHT and EQ being somewhat more effective than DMF and DIS. A similar order of activity was recorded in the relative ability of these substances to increase hepatic activities of DT-diaphorase, consistent with a role for this enzyme in facilitating conjugation and excretion of this naphthoquinone. In contrast, all the compounds increased the haemolytic activity of 2-hydroxy-1,4-naphthoquinone. DMF and DIS were significantly more effective in this regard than BHA, BHT and EQ. DMF and DIS also caused a much greater increase in levels of DT-diaphorase in the intestine, suggesting that 2-hydroxy-1,4-naphthoquinone is activated by this enzyme in the gut. BHA, BHT and EQ had no effect on the nephrotoxicity of 2-hydroxy-1,4-naphthoquinone, but the severity of the renal lesions was decreased in rats pre-treated with DMF and DIS. The results of the present experiments show that modulation of tissue levels of DT-diaphorase may not only alter the severity of naphthoquinone toxicity in vivo, but may also change the relative toxicity of these substances to different target organs.

Toxicity of 2,3-dialkyl-1,4-naphthoquinones in rats: Comparison with cytotoxicity in vitro

The short-term toxicities of 2-methyl-1,4-naphthoquinone and a series of 2,3-dialkyl-1,4-naphthoquinones have been determined in rats and compared with their ability to cause oxidative damage to erythrocytes in vitro. In accord with previous results, 2-methyl-1,4-naphthoquinone caused marked oxidative damage to erythrocytes in vitro and haemolytic anaemia in rats. The dialkylnaphthoquinones were also haemolytic agents in vivo, with 2,3-dimethyl-1,4-naphthoquinone being particularly active. Unlike the monoalkyl derivative, however, these substances caused little or no damage to red cells in vitro. The in vivo toxicity of dialkylnaphthoquinones cannot, therefore, be predicted on the basis of in vitro cytotoxicity tests.

Effects and mechanisms of action of the ergopeptides ergotamine and ergovaline and the effects of peramine on reticulum motility of sheep

OBJECTIVE To investigate effects and mechanisms of ergotamine and ergovaline and effects of peramine on reticulum motility of sheep. SAMPLE POPULATION 3 sheep with indwelling electrodes in the reticulum and samples of reticulum collected from 126 sheep at an abattoir. PROCEDURES In conscious sheep, motility was recorded as integrated electromyograms from the reticulum. Ergotamine was administered IV alone or in combination with the cholinergic muscarinic receptor antagonist atropine to sheep, and motility of the reticulum was assessed. In vitro, whole wall strips of the reticulum, cut in a direction to record longitudinal muscle activity via force transducers, were placed in 10-mL organ baths and superfused with Tyrode Ringers solution at 37 degrees C and oxygenated with 95% oxygen and 5% carbon dioxide. Testing involved incubation of reticulum strips with ergotamine, ergovaline, and peramine and measurement of motility of the reticulum tissues. RESULTS Administration of ergotamine to sheep reduced the frequency of reticulum contractions and increased baseline electromyographic activity (tonus). Frequency was unaffected by atropine, whereas tonus was significantly reduced. In vitro, ergotamine and ergovaline increased tonic contractions and stimulated phasic contractions of reticulum tissues and potentiated electrically stimulated contractions. Atropine and tetrodotoxin reduced tonic contractions, but stimulation of large-amplitude phasic contractions remained. Peramine had no effect on motility of reticulum tissues. CONCLUSIONS AND CLINICAL RELEVANCE Results of the study indicated that peripheral excitatory effects of the ergopeptides on motility of the reticulum appear to be mediated partly through myenteric neurons and muscarinic receptors and also through direct effects on the muscles.

Image processing of hematoxylin and eosin-stained tissues for pathological evaluation.

Color and geometric characteristics of stained areas in histochemical slides are among the features pathologists assess to evaluate the severity of lesions. In this research, image processing techniques were used to perform objective quantification of these characteristics in images of H&E-stained spleen tissues. A segmentation algorithm was developed to isolate the areas of interest in microscopic tissue images. Image features important to pathological evaluation were then extracted. These features were used to build statistical and neural network models to predict pathologist scores. A linear regression model predicted the scores to an R2-value of 0.6, and a neural network model classified samples to an accuracy of 75%. The results show the usefulness of image processing as a tool for pathological evaluation.

Effects of modulation of tissue activities of DT-diaphorase on the toxicity of 2,3-dimethyl-1,4-naphthoquinone to rats

The enzyme DT-diaphorase mediates the two-electron reduction of quinones to hydroquinones. It has previously been shown that the toxicity of 2-methyl-1,4-naphthoquinone to rats is decreased by pre-treatment of the animals with compounds that increase tissue levels of this enzyme. In contrast, the severity of the haemolytic anaemia induced in rats by 2-hydroxy-1,4-naphthoquinone was increased in animals with high levels of DT-diaphorase. In the present experiments, the effect of alterations in tissue diaphorase activities on the toxicity of a third naphthoquinone derivative, 2,3-dimethyl-1,4-naphthoquinone, has been investigated. This compound induced severe haemolysis and slight renal tubular necrosis in control rats. Pre-treatment of the animals with BHA, a potent inducer of DT-diaphorase, diminished the severity of the haemolysis induced by this compound and abolished its nephrotoxicity. Pre-treatment with dicoumarol, an inhibitor of this enzyme, caused only a slight increase in the haemolysis induced by 2,3-dimethyl-1,4-naphthoquinone, but provoked a massive increase in its nephrotoxicity. Modulation of DT-diaphorase activity in animals may therefore not only alter the severity of naphthoquinone toxicity, but also cause pronounced changes in the site of toxic action of these substances. The factors that may control whether induction of DT-diaphorase in animals will decrease or increase naphthoquinone toxicity are discussed.