Hepatic xenobiotic metabolism of cylindrospermopsin in vivo in the mouse.

Cylindrospermopsin (CYN) is a hepatotoxin isolated from the blue-green alga Cylindrospermopsis raciborskii. The role of both glutathione (GSH) and the cytochrome P450 enzyme system (P450) in the mechanism of toxicity of CYN has been previously investigated in in vitro systems. We have investigated the role of GSH and P450 in vivo in mice. Mice pre-treated with buthionine sulphoximine and diethyl maleate to deplete hepatic GSH prior to dosing with 0.2mg/kg CYN showed a seven-day survival rate of 5/13 while the control group rate was 9/14. Dosing mice with 0.2mg/kg CYN produced a small decrease in hepatic GSH with a characteristic rebound effect at 24h. The magnitude of this effect is however small and combined with the non-significant difference in survival rates after GSH depletion suggest depletion of GSH by CYN could not be a primary mechanism for CYN toxicity. Conversely, pre-treatment with piperonyl butoxide, a P450 inhibitor, protected mice against CYN toxicity giving a survival rate of 10/10 compared with 4/10 in the control group (p < 0.05 Chi squared) and was protective at doses up to 0.8 mg/kg, suggesting activation of CYN by P450 is of primary importance in the mechanism of action.

Toxicity and uptake mechanism of cylindrospermopsin and lophyrotomin in primary rat hepatocytes.

The toxicities and uptake mechanisms of two hepatotoxins, namely cylindrospermopsin and lophyrotomin, were investigated on primary rat hepatocytes by using microcystin-LR (a well-known hepatotoxin produced by cyanobacteria) as a comparison. Isolated rat hepatocytes were incubated with different concentrations of hepatotoxins for 0, 24, 48 and 72 h. The cell viability was assayed by the tetrazolium-based (MTT) assay. Microcystin-LR, cylindrospermopsin and lophyrotomin all exhibited toxic effects on the primary rat hepatocytes with 72-h LC(50) of 8, 40 and 560 ng/ml, respectively. The involvement of the bile acid transport system in the hepatotoxin-induced toxicities was tested in the presence of two bile acids, cholate and taurocholate. Results showed that the bile acid transport system was responsible for the uptake, and facilitated the subsequent toxicities of lophyrotomin on hepatocytes. This occurred to a much lesser extent with cylindrospermopsin. With its smaller molecular weight, passive diffusion might be one of the possible mechanisms for cylindrospermopsin uptake into hepatocytes. This was supported by incubating a permanent cell line, KB (devoid of bile acid transport system), with cylindrospermopsin which showed cytotoxic effects. No inhibition of protein phosphatase 2A by cylindrospermopsin or lophyrotomin was found. This indicated that other toxic mechanisms besides protein phosphatase inhibition were producing the toxicities of cylindrospermopsin and lophyrotomin, and that they were unlikely to be potential tumor promoters.

A sensitive and specific assay for glutathione with potential application to glutathione disulphide, using high-performance liquid chromatography-tandem mass spectrometry.

We have utilised the combination of sensitivity and specificity afforded by coupling high-performance liquid chromatography (HPLC) to a tandem mass spectrometer (MS-MS) to produce an assay which is suitable for assaying glutathione (GSH) concentrations in liver tissue. The sensitivity suggests it may also be suitable for extrahepatic tissues. The method has been validated for GSH using mouse liver samples and also allows the assay of GSSG. The stability of GSH under conditions relevant to the assay has been determined. A 20-microl amount of a diluted methanol extract of tissue is injected with detection limits of 0.2 pmol for GSH and 2 pmol for GSSG. The HPLC uses an Altima C18 (150 x 4.6 mm, 5 microm) column at 35 degrees C. Chromatography utilises a linear gradient from 0 to 10% methanol in 0.1% formic acid over 5 min, with a final isocratic stage holding at 10% methanol for 5 min. Total flow rate is 0.8 ml/min. The transition from the M+H ion (308.1 m/z for GSH, and 613.3 m/z for GSSG) to the 162.0 m/z (GSH) and 355.3 m/z (GSSG) fragments are monitored.

Isolation and identification of the toxic peptides from Lophyrotoma zonalis (Pergidae) sawfly larvae.

The broad-leaved paper bark tree Melaleuca quinquenervia (Cav) (Myrtaceae) was introduced into Florida (USA) early in this century it has proliferated to such an extent that urgent measures are now required to control it. The sawfly Lophyrotoma zonalis (Pergidae) has been introduced as a possible biological control agent due to its ability to defoliate M. quinquenervia. Because toxic D-amino acid- containing peptides have been isolated from some sawfly species, L. zonalis larvae were processed using the previously reported method for the recovery of these compounds. The toxins lophyrotomin (as the free C-terminal acid) and a mixture of pergidin and Val (4)-pergidin were isolated at 0.36 and 0.43% yield of the dried larvae, respectively. Both compounds when dosed intraperitoneally to C57/Bl6 male mice were hepatotoxic with lowest lethal doses of 8 and 32 mg/kg, respectively. The pathology of the liver was different for each compound, with the lophyrotomin free acid causing a periportal haemorrhagic necrosis and the pergidin causing a periacinar coagulative necrosis.

Pyrrolizidine alkaloids in human diet.

Pyrrolizidine alkaloids are the leading plant toxins associated with disease in humans and animals. Upon ingestion, metabolic activation in liver converts the parent compounds into highly reactive electrophiles capable of reacting with cellular macromolecules forming adducts which may initiate acute or chronic toxicity. The pyrrolizidine alkaloids present a serious health risk to human populations that may be exposed to them through contamination of foodstuffs or when plants containing them are consumed as medicinal herbs. Some pyrrolizidine alkaloids (PA) adducts are persistent in animal tissue and the metabolites may be re-released and cause damage long after the initial period of ingestion. PAs are also known to act as teratogens and abortifacients. Chronic ingestion of plants containing PAs has also led to cancer in experimental animals and metabolites of several PAs have been shown to be mutagenic in the Salmonella typhimurium/mammalian microsome system. However, no clinical association has yet been found between human cancer and exposure to PAs. Based on the extensive reports on the outcome of human exposure available in the literature, we conclude that while humans face the risk of veno-occlusive disease and childhood cirrhosis PAs are not carcinogenic to humans.

Unique toxic peptides isolated from sawfly larvae in three continents.

D-Amino acid containing peptides have been found to be responsible for sawfly larvae poisoning in many parts of the world. These compounds, unique in the animal kingdom, were isolated from three different species of sawfly indigenous to Australia, Denmark and South America. The octapeptide, lophyrotomin, is the major toxin in the Australian and Danish species and is present in small amounts in the South American sawfly. Pergidin, the main toxin in the South American sawfly, is a heptapeptide containing a phosphoseryl residue. This, as far as we are aware, is the first example of such a peptide to be isolated from an animal source. Small amounts of pergidin have been found in the other two species. All available evidence suggests that both peptides are biosynthesised 'de novo' possibly as a protective device, however it cannot be excluded that microorganisms may be responsible. These compounds are stable to enzymatic breakdown because of their configuration and their strong chemical bonding and lipophilic character provide a potential for residues to remain in the host animal and cause significant changes.

H-ras activation is an early event in the ptaquiloside-induced carcinogenesis: comparison of acute and chronic toxicity in rats.

Bracken fern (Pteridium spp.) produces cancer of the urinary bladder and oesophagus in grazing animals and is a suspected human carcinogen. The carcinogenic principle ptaquiloside (PT), when activated to a dienone (APT), forms DNA adducts which eventually leads to tumor. Two groups of female Sprague-Dawley rats were given a chronic dose of 3 mg APT weekly for 10 weeks either by intravenous (i.v.) tail vein or by intragastric (i.g.) route. A third group was given a weekly dose of 6 mg of APT for 3 weeks by the i.g. route corresponding to acute dosing. Both chronic i.v. and i.g. dosed animals showed ischemic tubular necrosis in the kidney but only i.v. dosed animals developed adenocarcinomas of the mammary glands. Acutely dosed i.g. animals produced apoptotic bodies in the liver, necrosis of blood cell precursors in the bone marrow and ischemic tubular necrosis in the kidney but they did not develop tumors. No mutations were found in the H-ras and p53 genes in the mammary glands of either the i.g. rats or the tumor-bearing i.v. rats. However, the mammary glands of a fourth group of rats, which received APT by i.v. and killed before tumor development, carried Pu to Pu and Pu to Py double mutations in codons 58 and 59 of H-ras. This study indicates that the route of administration plays a role in the nature of the disease expression from ptaquiloside exposure. In addition to confirming the role of APT in the PT-induced carcinogenesis our finding suggests that activation of H-ras is an early event in the PT-carcinogenesis model.

Bracken fern carcinogenesis: multiple intravenous doses of activated ptaquiloside induce DNA adducts, monocytosis, increased TNF alpha levels, and mammary gland carcinoma in rats.

AIMS: (1) establish a rat model for investigating ptaquiloside (PT) carcinogenesis via intravenous dosing; (2) determine the role of activated PT (APT) in this model; and (3) monitor changes at molecular (DNA adducts, TNF alpha levels) and cellular (histopathology) levels. METHODS: Sprague-Dawley rats were dosed with PT or APT intravenously for 10 consecutive weeks. One group of animals was sacrificed immediately for TNF alpha and DNA adduct analyses. A second group of animals was kept alive for 30 more weeks to allow for tumour formation. Tissues were collected at the end of the experiment for histopathological studies. RESULTS: Rats dosed with PT or APT showed marked increase in monocyte and TNF alpha levels. These levels remained high even 30 weeks after the last dosing. Analysis of DNA showed the presence of DNA adducts in APT-treated animals in target organs. In addition, 40% of APT-treated rats developed mammary gland carcinomas. CONCLUSION: This is the first study to demonstrate the potential of activated PT as a carcinogen in vivo. In addition, our findings suggest that PT exposure can be monitored using monocyte and TNF alpha levels.

Results of anterior transposition of the inferior oblique.

PURPOSE: We present the results of anterior transposition of the inferior oblique in a series of patients with inferior oblique overaction and dissociated vertical deviation (DVD). PATIENTS AND METHODS: We performed a retrospective study of 37 procedures on 21 patients who had unilateral or bilateral inferior oblique anterior transpositions. Before surgery, patients had +1 to +3 inferior oblique overaction and +1 (< 10 PD) or +2 (10 PD-20 PD) degree of DVD. The inferior oblique insertion was transposed to between 2 mm posterior to and 2 mm anterior to the temporal border of the inferior rectus insertion. Mean follow-up period was 27 months. RESULTS: Incidence of inferior oblique overaction of +2 or more was reduced from 84% before surgery to 16% at last postoperative assessments. Some 43% of eyes had no inferior oblique overaction and 86% had an improvement in the degree of inferior oblique overaction. At last assessments, 57% of eyes had no evidence of DVD and 68% of eyes had no evidence of DVD or an improvement in the degree of DVD. No patient who had unilateral anterior transposition developed hypotropia in primary position and there was no evidence of inferior oblique underaction in any patient at last assessment. Three patients requiring repeat inferior oblique surgery are discussed, including one patient who developed a large Y-pattern exotropia after bilateral anterior transposition of the inferior obliques. CONCLUSIONS: Inferior oblique anterior transposition has a place in the treatment of coexistent inferior oblique overaction and dissociated vertical deviation.

Coexistent orbital and cerebellar venous anomalies in linear sebaceous naevus syndrome.

BACKGROUND: Orbital venous anomalies can result in significant morbidity and have been reported in association with other venous anomalies, some with the potential for serious complication. METHODS/RESULTS: We present a case of an orbital venous anomaly coexistent with a large cerebellar venous angioma and a linear sebaceous naevus. Clinical features, associations, complications and management principles are presented. CONCLUSION: Upon clinical recognition of an orbital venous anomaly, brain imaging and appropriate clinical assessment should be considered in light of the possibility of coexistence of potentially life-threatening lesions.

Bone marrow and renal injury associated with haloalkene cysteine conjugates in calves.

Almost 40 years ago, it was reported that cattle-feed which had been extracted with hot trichloroethylene and then fed to calves produced renal injury and a fatal aplastic anaemia. The toxic factor was subsequently identified as S-(1,2-dichlorovinyl)-L-cysteine (DCVC). These original findings have been confirmed, a single intravenous dose of DCVC at 4 mg/kg, or 0.4 mg/kg intravenously per day administered for 10 days to calves produced aplastic anaemia, and renal injury after a single dose of 4 mg/kg. The toxicity to calves of a number of other haloalkene cysteine conjugates has been examined to ascertain whether, like DCVC, they produce bone marrow and renal injury. Intravenous administration of the N-acetyl cysteine conjugate of DCVC produced renal but not bone marrow injury at a molar equivalent dose to DCVC, indicating that the calf can deacetylate the mercapturic acid and further that sufficient chemical had reached the kidney to be a substrate for the enzyme cysteine conjugate beta-lyase. However, intravenous administration of the alpha-methyl analogue of DCVC, which cannot undergo metabolism via the enzyme cysteine conjugate beta-lyase, was without toxicity at doses about five-fold higher than DCVC. These latter findings provide strong evidence that metabolism of DCVC via the enzyme beta-lyase is necessary for bone marrow and renal injury to occur. The cysteine conjugates of perchloroethylene and hexachloro-1,3-butadiene(HCBD) when given intravenously to calves at molar equivalent doses to DCVC, or above, did not produce either bone marrow or renal injury. In contrast, intravenous administration of the cysteine conjugate of tetrafluoroethylene (TFEC) produced severe renal tubular injury in calves without affecting the bone marrow. In vitro studies with these haloalkene cysteine conjugates showed, like DCVC, that they were good substrates for calf renal cysteine conjugate beta-lyase and toxic to renal cells as judged by their ability to reduce organic anion and cation transport by slices of calf renal cortex and inhibit the renal enzyme glutathione reductase. Calves were also dosed either orally or intravenously with HCBD to assess its toxicity. HCBD at higher molar equivalent doses than DCVC produced mid-zonal necrosis in the liver, renal tubular necrosis but no bone marrow injury in calves. The key findings emerging from these studies are (1) that none of the other cysteine conjugates, at molar equivalent doses to DCVC and above, produce bone marrow injury in calves, (2) TFEC produced only renal injury, suggesting that sufficient of the other conjugates had not reached the kidney for metabolism by beta-lyase to produce cytotoxicity and (3) that HCBD itself is more toxic than its cysteine or mercapturic acid conjugate, suggesting that pharmaco-kinetics and disposition are important factors in determining the toxicity of these conjugates to calves. Further studies are needed to understand the basis for the selective toxicity of DCVC to the bone marrow of calves.

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 fem-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.

Bracken fern (Pteridium spp.) carcinogenicity and human health--a brief review.

Bracken fern (Pteridium spp.), one of the most abundant plants on the planet, is well known to cause cancer naturally in sheep and cattle. It contains, in some locations, extremely high concentrations of ptaquiloside which almost certainly is its major carcinogen. Ptaquiloside is transferred through milk. There is epidemiological evidence that the bracken carcinogen, in special situations, may cause cancer in man. Ptaquiloside and its animal models of carcinogenesis also offer good tools for the study of cancer.

Directly toxic effects of plant chemicals which may occur in human and animal foods.

Pyrrolizidine alkaloids are among the most significant plant chemicals causing disease in animals and humans. After absorption from the gut, the compounds are converted to electrophilic pyrroles in the liver which, apart from causing damage to this organ, may escape to cause injury to extraheptic tissues such as the lungs, heart, and kidneys. A group of compounds more recently found to be associated with neurotoxicity are various polyhydroxyalkaloids which are able to interfere with polysaccharide metabolism. They are able to inhibit lysosomal monosaccharidases by virtue of their structural resemblance to the transition state of particular sugar molecules. The resulting lysosomal storage diseases have pathology identical to that of the respective congenital and heritable lysosomal storage diseases which occur in animals and humans. Consumption of cycad plants by cattle may cause a neurotoxicity characterised mainly by a posterior sensory ataxia. In recent years, cycads are considered to be a risk factor for a spectrum of progressive neuro degenerative diseases of humans in the Western Pacific region. The known toxins in the plant are the methylazoxymethanol glycosides which are hepatotoxic and carcinogenic, and the neurotoxic non-protein amino acid beta-methylaminoalanine. A plant carcinogen which can be of great abundance in the nutritional environment is the illudine norsesquiterpene glucoside ptaquiloside which is found in bracken fern. This is the only plant carcinogen which causes natural outbreaks of bladder and/or intestinal cancer in livestock. Many legumes contain phytooestrogens, notably isoflavones. Consumption of these compounds at high levels by sheep can cause extensive lesions of the genitalia of females and castrated males.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation of a compound from Eupatorium adenophorum (Spreng.) [Ageratina adenophora (Spreng.)] causing hepatotoxicity in mice.

Regular ingestion of Eupatorium adenophorum [Ageratina adenophora (Spreng.)] or Crofton weed causes chronic pulmonary disease in horses mainly in Australia, New Zealand, and the Himalayas. The disease is characterized by pulmonary interstitial fibrosis, emphysema, alveolar epithelisation and reduced tolerance to exercise. Horses apparently are the only animals affected and there are numerous reports of farms losing all their horses. The disorder was produced experimentally in horse feeding trials, and it was shown that characteristic lesions occurred in the lungs. In studies with laboratory animals, mice were shown to be suitable test animals, but in this species lesions occur in the liver rather than the lungs. The hepatic injury in these animals is characterized by multiple areas of focal necrosis of the parenchyma associated with degeneration and loss of the epithelium lining the small bile ducts. The active principle 9-oxo-10,11 dehydroagerophorone responsible for these lesions in mice has been isolated from E. adenophorum. Although the compound has been shown to exhibit toxicity to larvae of invertebrate species, no mammalian toxicity studies have been previously reported involving the isolated toxin. The mechanism of the toxic effect of the compound as well as its possible relevance to the respiratory disease in the horse remain to be investigated.

Concentration of ptaquiloside, a major carcinogen in bracken fern (Pteridium spp.), from eastern Australia and from a cultivated worldwide collection held in Sydney, Australia.

Two surveys of bracken fern for the concentration of the carcinogen ptaquiloside (PT) have been carried out, one of bracken fern from the eastern side of Australia and the other from a worldwide collection of bracken clones held in Sydney Australia. Bracken from eastern Australia contained concentrations of ptaquiloside ranging from 0 to 12,945 micrograms PT/g. From 91 samples 15% contained greater than 5,000 micrograms PT/g and 57% of samples contained more than 1,000 micrograms PT/g bracken on the dry weight basis. Ptaquiloside concentrations were highest in Pteridium revolutum and from P. esculentum from areas where bovine enzootic haematuria was known to occur. Bracken from the cultivated bracken clone collection from world-wide sources tended to have lower concentrations of ptaquiloside ranging from 0 to 9,776 micrograms PT/g. From 77 samples, 8% contained more than 5,000 micrograms PT/g and 35% contained more than 1,000 micrograms PT/g bracken. Samples from both the eastern Australia survey and the Australian representatives in the worldwide collection showed significantly higher concentrations of PT in the P. esculentum collected from the more southern states. In samples from the worldwide collection there were no statistical differences in the concentrations of PT in bracken between taxa.

A comparison of the effects of aflatoxin B1 on the livers of rats and duck hepatitis B virus-infected and noninfected ducks.

A need exists for an appropriate animal model for the involvement of both hepatitis B virus infection and ingestion of aflatoxins in the etiology of liver cancer. Duck hepatitis B virus-infected ducks, on the basis of hepatoma development in the wild in China, appear to offer this possibility. The duck has been reexamined as a model system, and key metabolic processes have been assayed in comparison with the rat model for hepatocarcinogenesis. Aflatoxin B1 was found to be more actively metabolized by hepatic microsomes isolated from Pekin ducks in vitro to the aflatoxin B1-8,9-epoxide than corresponding fractions from the rat, and in vivo, higher levels of aflatoxin B1-guanine adduct were formed in hepatic DNA than in the livers of the aflatoxin B1-sensitive F344 rat. Repair of this DNA lesion in the duck and the subsequent formation of the ring-opened aflatoxin B1-FAPy adduct paralleled that in the rat. No effect of duck hepatitis B virus infection was found on any of these biochemical processes. The formation of hepatic lesions was also studied, and lesions were compared with those seen in the aflatoxin B1-treated rat. Histological analysis of necropsy specimens from ducks, 20 mo after the ducks received doses of aflatoxin B1 (25 and 50 micrograms/kg body wt), showed almost complete regression of the early acute lesions, with no evidence of neoplasia. Male F344 rats treated with aflatoxin B1 150 micrograms/kg 5 days/wk for 4 wk had extensive bile duct hyperplasia at the end of the treatment period and 100% incidence of hepatocellular carcinoma after 52 wk. The possible basis for the relative sensitivity of ducks and rats to the carcinogenic action of aflatoxin B1 is discussed.

Some effects of chronic mercuric chloride intoxication on renal function in a horse.

Chronic mercuric chloride intoxication in an aged horse given 0.8 mg Hg/kg/day for 14 weeks was manifest by signs of progressive respiratory difficulty and renal disease. The effects were not self-limiting after mercury was withdrawn, and the animal was destroyed six weeks later. Renal function changes included heavy glycosuria, modest proteinuria, phosphaturia, reduced urine osmolality, gradually increasing urine production, reduced glomerular filtration rate, and terminally, azotemia. The condition bore similarities to the Fanconi syndrome in man. Urinary gamma-glutamyl transpeptidase, alkaline phosphatase and amino-aspartate transferase activities were inconsistent indicators of tubular damage in random samples at this dose rate. The pathologic response was characterized by extensive granulomatous infiltration throughout the lungs, in particular, and to a lesser extent in the kidneys, liver and bone marrow. The renal changes included this marked interstitial reaction and proximal tubular degeneration. Mercury levels were negligible in the lungs and highest in the renal cortex. The granulomatous reaction was not encountered in previous mercury toxicity studies in horses and may indicate an individual sensitivity to the agent.

Selective injury to rat liver Kupffer cells caused by beryllium phosphate: an explanation of reticuloendothelial blockade.

The i.v. administration of suspensions of beryllium phosphate (5-50 mumol/kg) to rats resulted in the vacuolation of hepatic Kupffer cells within 3 h. After 6 h necrotic Kupffer cells were common throughout the sinusoids of the liver but no changes were detected in the hepatic parenchymal cells during this period. A significant reduction in the numbers of intrasinusoidal cells was observed 14 h after treatment but this population had reverted to normal within 24 h. The administration of colloidal carbon to treated animals at this time did, however, demonstrate a reduction in the complement of functional endocytic cells. These results demonstrate a selective destruction of endocytic cells in the liver by this particulate toxin and the limited response by the organ to this injury. These observations are the most probable explanation for the reticuloendothelial blockade known to be caused in vivo by beryllium phosphate.