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.

Extraction and purification of the zwitterions cylindrospermopsin and deoxycylindrospermopsin from Cylindrospermopsis raciborskii.

The hepatotoxin cylindrospermopsin (CYN) has been isolated from the cyanobacterium Cylindrospermopsis raciborskii (C. raci.). Efforts to study this toxin have been hampered by the time-consuming requirement to extract it from cultures of the organism. It is usually extracted from lyophilized cells collected from a laboratory culture. Our preliminary work suggested far more of the toxin is available in solution in the culture media than in the cells collected. We have therefore investigated the use of commercially available solid phase extraction sorbents to extract CYN from culture media in which C. raci. has been grown. A range of reverse phase and ion-exchange sorbents were tested across a range of pHs for their ability to retain CYN without success. Subsequently, graphitized carbon cartridges were found to retain CYN strongly. Elution with 5% formic acid in methanol allowed the CYN to be regained for final purification by HPLC. Deoxy-CYN, an analog of CYN can also be extracted using this procedure.

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.

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.

Distribution of 14C cylindrospermopsin in vivo in the mouse.

Radiolabelled 14C cylindrospermopsin (CYN) has been prepared and used to investigate the distribution and excretion of CYN in vivo in male Quackenbush mice. At a dose of 0.2 mg/kg (i.e., approx. median lethal dose) the following mean (SD) urinary and faecal recoveries (cumulative) were obtained, respectively: (0-6 hours, n = 4) 48.2 (29.3)%, 11.9 (21.4)%; (0-12 hours, n = 12) 66.0 (27.1)%, 5.7 (5.6)%; (0-24 hours, n = 12) 68.4 (26.7)%, 8.5 (8.1)%. Mean (SD) recoveries from livers at 6 hours were 20.6 (6.4)% (n = 4), at 48 hours 13.1 (7.7)% (n = 8), and 5-7 days were 2.1 (2.1)% (n = 8). A substantial amount (up to 23%) can be retained in the liver for up to 48 hours with a lesser amount retained in the kidneys. The excretion patterns show substantial interindividual variability between predominantly faecal or urinary excretion, but these patterns are not related in any simple manner to the outcome in terms of toxicity. There is at least one methanol-extractable metabolite as well as a nonmethanol-extractable metabolite in the liver. The methanol-extractable metabolite was not found in the kidney and is more hydrophilic than CYN itself on reverse phase.

Cylindrospermopsin, a cyanobacterial alkaloid: evaluation of its toxicologic activity.

This paper describes the natural occurrence of the toxin, cylindrospermopsin, in two species of cyanobacteria found in Australia. The structure and chemical properties of this compound are described along with a nontoxic analog of cylindrospermopsin. The results of both intraperitoneal (IP) and oral dosing of mice show that hepatotoxicity is the main effect of cylindrospermopsin in vivo, but that a thrombohemorrhagic phenomenon is observed in a proportion of dosed animals. It has been shown that the toxin can be metabolized in vivo and that a bound metabolite occurs in the liver. Cytotoxicity experiments using cell cultures show that cylindrospermopsin is more cytotoxic to isolated rat liver hepatocytes than to other cell types. Risk assessment calculations show that guideline values for cylindrospernopsin in drinking water should lie in the low microgram per liter range.

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.

Dehydromonocrotaline generates sequence-selective N-7 guanine alkylation and heat and alkali stable multiple fragment DNA crosslinks.

Monocrotaline is a pyrrolizidine alkaloid known to cause toxicity in humans and animals. Its mechanism of biological action is still unclear although DNA crosslinking has been suggested to a play a role in its activity. In this study we found that an active metabolite of monocrotaline, dehydromonocrotaline (DHM), alkylates guanines at the N7 position of DNA with a preference for 5'-GG and 5'-GA sequences. In addition, it generates piperidine- and heat-resistant multiple DNA crosslinks, as confirmed by electrophoresis and electron microscopy. On the basis of these findings, we propose that DHM undergoes rapid polymerization to a structure which is able to crosslink several fragments of DNA.

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.

Results of treatment of superior oblique overaction by silicone tendon-expander technique.

PURPOSE: To report the results of treatment of a series of patients with superior oblique overaction using the superior oblique silicone tendon-expander technique. METHODS: A chart review of 17 patients with superior oblique overaction who had a total of 26 silicone tendon-expander procedures was conducted. RESULTS: Mean preoperative degree of superior oblique overaction was +2.7. 92% of eyes had mild (+1) or no residual overaction at last postoperative assessment (follow-up range: 6 to 59 months). Of 15 patients with preoperative A-pattern of 10 prism diopter (delta) or more, only two patients (13%) had A-pattern of 10 delta or more at last assessment. Of 13 patients with preoperative hypotropia in primary position, five patients (38%) had no vertical deviation in primary position, and seven patients (54%) had persistent, but less vertical deviation in primary position at last assessment (mean reduced from 11 delta to 4 delta). No patient manifested superior oblique palsy at their last postoperative assessment. CONCLUSIONS: We believe that the superior oblique tendon-expander technique should be strongly considered for the treatment of superior oblique overaction associated with A-pattern or hypotropia in primary position, because it has a high success rate and a low incidence of postoperative complications. Consecutive superior oblique underaction did not occur in this series.

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.

Intravitreal cilia in phakic penetrating eye injury.

BACKGROUND: Intraocular cilia present clinical perplexity due to their radiolucency, the extremely variable ocular response to such cilia, and the inadvisability of using MRI in cases of suspected metallic intraocular foreign bodies (IOFB). METHODS: Two cases of intravitreal cilia associated with phakic penetrating eye injury are described where preoperative CT scan revealed no retained IOFB. RESULTS: B-scan ultrasonography detected intravitreal cilia in one patient and raised this suspicion in the other. One patient presented with endophthalmitis unresponsive to intravitreal antibiotics, the other with culture-negative anterior uveitis. Both underwent vitrectomy and removal of cilia. CONCLUSIONS: Intravitreal cilia should be considered in penetrating eye injuries even in phakic eyes with no radiological evidence of IOFB, especially if associated with endophthalmitis. B-scan ultrasonography may aid detection of intravitreal cilia and thus alter clinical management.

Isolation and characterisation of urushiol components from the Australian native cashew (Semecarpus australiensis).

Native cashew (Semecarpus australiensis) is a well-known food source for aboriginal people of northeastern Queensland and the Northern Territory. It is also well known that contact with the seeds at a certain stage of growth can cause sever dermatitis in susceptible individuals. To prepare the fruits for eating, they are commonly treated by leaching for 2-7 days with water followed by heating in bark, and this treatment apparently produces an edible cashew nut. Recently, attempts have been made to use this valuable source of food by a commercial company. It was necessary to identify the active principle(s) in the seeds to determine the most effective way of rendering the seed suitable for human consumption without altering the flavour. By using solvent extraction and silica-gel chromatography, a fraction containing one major urushiol was obtained. Its structure was confirmed by comparison of its NMR and mass spectral (MS) data with that previously reported. This compound, which is found in high yield, is also found in poison ivy (Toxiodenron radicans) and is responsible for dermatitis in susceptible individuals. A method of removing the active principle from the seed has been suggested.

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.

Macula toxicity after intravitreal amikacin.

BACKGROUND: Although intravitreal aminoglycosides have substantially improved visual prognosis in endophthalmitis, macular infarction may impair full visual recovery. METHODS: We present a case of presumed amikacin retinal toxicity following treatment with amikacin and vancomycin for alpha-haemolytic streptococcal endophthalmitis. RESULTS: Endophthalmitis resolved with improvement in visual acuity to 6/24 at three months. Fundus fluorescein angiography confirmed macular capillary closure and telangiectasis. CONCLUSIONS: Currently accepted intravitreal antibiotic regimens may cause retinal toxicity and macular ischaemia. Treatment strategies aimed at avoiding retinal toxicity are discussed.

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.

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.