The GreenScreen genotoxicity assay: a screening validation programme.

A yeast (Saccharomyces cerevisiae) DNA repair reporter assay termed the GreenScreen assay (GSA) is described. This is a novel, cost-effective genotoxicity screen, developed to provide a pre-regulatory screening assay for use by the pharmaceutical industry and in other applications where significant numbers of compounds need to be tested. It provides a higher throughput and a lower compound consumption than existing eukaryotic genotoxicity assays and is sensitive to a broad spectrum of mutagens and, importantly, clastogens. We describe a simple, robust assay protocol and a validation study. The end-point of the test reflects the typically eukaryotic chromosomes and DNA metabolizing enzymes of yeast. The capacity for metabolic activation (MA) in yeast is limited compared with the mammalian liver or its extracts, but the assay does detect a subset of compounds that would require MA in existing genotoxicity tests. The GSA detects a different spectrum of compounds to bacterial genotoxicity assays and thus, together with an in silico structure-activity relationship (SAR) screen, and possibly a high throughput bacterial screen, would provide an effective preview of the regulatory battery of genotoxicity tests.

trans-Nonachlor and cis-nonachlor toxicity in Sprague-Dawley rats: comparison with technical chlordane.

cis-Nonachlor and trans-nonachlor are bioaccumulating components of the pesticide chlordane, which can be detected in various environmental biota and in humans. Existing studies have focused on the potential adverse health effects of the parent chlordane mixture. Comparable toxicity data are nonexistent for individual chlordane constituents such as trans-nonachlor, cis-nonachlor, or oxychlordane, which are among the most common chlordane-related environmental contaminants and tissue residues. In this study, rats were administered cis-nonachlor, trans-nonachlor, or technical chlordane by gavage for 28 days at doses of 0.25 to 25 mg/kg body weight. Residue analyses indicated that trans-nonachlor accumulation in adipose was greater than cis-nonachlor when rats were administered each chemical under identical conditions of dose and exposure. For all test chemicals, the major metabolite oxychlordane accumulated in adipose tissue. Adipose tissue residue levels of all test chemicals and the major metabolite were higher in female rats. The liver was a target organ in male and female rats, indicated by increased liver weight and histopathological changes consistent with microsomal enzyme induction. Hepatic changes were most pronounced in rats treated with trans-nonachlor. Elevated kidney weights and depressed organic ion transport were observed in males treated with trans-nonachlor and chlordane. Although in general, changes in target organs and clinical chemistry endpoints were similar for all 3 test chemicals, the approximate toxicity ranking from most to least toxic was trans-nonachlor > technical chlordane > cis-nonachlor.

A comparison of clinical, histopathological and cell-cycle markers in rats receiving the fungal toxins fumonisin B1 or fumonisin B2 by intraperitoneal injection.

Fumonisins B1 and B2 (FB1 and FB2) are fungal secondary metabolites produced by members of the genus Fusarium. Although FB1 is usually detected in greater quantities, FB2 frequently co-occurs in contaminated feeds and foods and contributes to the total toxin load. In the present study, the comparative toxicity of FB1 and FB2 was examined in male Sprague-Dawley rats administered toxin (0.75 mg/kg body weight) or vehicle control intraperitoneally (ip) for 2, 4 or 6 consecutive days. Clinical changes, including elevated serum cholesterol, alanine aminotransferase (ALT), creatinine and protein, were slightly more pronounced in FB1-treated rats. The most consistent hematological change was an increase in vacuolated bone marrow cells, which was more pronounced in FB1-treated rats. Histopathological changes were similar in FB1- and FB2-treated rats and included single cell necrosis in kidneys and liver, cytoplasmic vacuolation in adrenal cortex and lymphocytolysis in thymus. In the liver mRNA expression for the cyclin kinase inhibitor p21 gene was significantly increased in FB1- and FB2-treated rats, compared to controls. Expression of mRNA for the cyclin D1 gene was significantly depressed in FB2-treated rats. Hepatic cyclin E mRNA was elevated in response to FB1 and FB2 compared to controls. In FB2-treated animals this corresponded with decreased liver p27 mRNA expression. Hepatic proliferating cell nuclear antigen (PCNA) transcription was elevated in FB1- but not FB2- treated rats. Changes in liver microsomal protein levels of p27, cyclin E and PCNA were similar to changes in gene expression. In contrast, cyclin D1 protein levels were elevated in rats treated with FB1 and, to a lesser extent, FB2. The data indicate that FB1 and FB2 can alter the expression of genes associated with the cell cycle, and indicate a need for a further understanding of the mechanistic basis of FB1 and FB2 toxicity.

Retrospective evaluation of serum ornithine carbamyltransferase activity as an index of hepatotoxicity in toxicological studies with rats.

The sensitivity of elevated serum ornithine carbamyltransferase (OCT) as an index of hepatotoxicity in rats was assessed in different studies conducted over a number of years and originally designed to examine the toxicity or carcinogenicity of a variety of test chemicals and diets. Changes in serum OCT activities were compared with the more widely used clinical endpoints, alanine aminotranserase (ALT) and aspartate aminotransferase (AST). In the first study, rats received a single oral dose of the hepatotoxic and hepatocarcinogenic fungal toxin aflatoxin B(1) (AFB(1)). The increase in enzyme levels between control and AFB(1)-treated rats was greater for serum OCT than for ALT or AST. This response was similar to the changes in serum enzyme levels in studies where rats ingested a hepatotoxic and hepatocarcinogenic choline deficient (CD) diet. When rats were exposed to the hepatotoxic and nephrotoxic fungal toxin fumonisin B(1) (FB(1)) by intraperitoneal injection for 6 days, serum AST and ALT were significantly elevated above control levels while OCT was unaffected. The peroxisome proliferator ciprofibrate caused elevated ALT and AST but not OCT at week 52 of dietary exposure, after the development of liver nodules and tumours. Of the two liver-specific enzymes examined in all of the studies, ALT was more consistently predictive of hepatotoxicity than OCT.

Effect of liana cutting on water potential and growth of adult Senna multijuga (Caesalpinioideae) trees in a Bolivian tropical forest.

Lianas, or woody climbing plants, are a major constituent of seasonally dry tropical forests, and are thought to impact negatively their host trees. In this study we evaluated whether liana presence was associated with reduced leaf water potentials and growth in adult Senna multijuga trees during the dry season in a lowland Bolivian forest. We used leaf water potentials in trees as a first approach to assess trees' water status, under the assumption that leaf water potentials become more negative when water losses (via transpiration) exceed gains (by uptake). We measured relative growth in girth at 1.5 m height (gbh) to quantify tree growth. At the beginning of the 1996 dry season (early June), we selected 20 S. multijuga trees 10-20 cm dbh, and measured their gbh. We also recorded pre-dawn and mid-day leaf water potentials in these trees. In ten experimental trees all lianas were then cut, while the remaining trees were used as controls. Pre-dawn and mid-day water potentials were re-measured 1 day after liana-cutting, and then every week in all trees for 1 month and then at 3 and 5 months, until the beginning of the next rainy season (November); gbh was measured again in July 1997 to estimate relative growth rate. Liana removal was associated with less negative pre-dawn (-0.3 vs -0.4 MPa) and mid-day (-0.5 vs -0.7 MPa) water potentials in trees during the dry season. This difference appeared as early as 1 day after cutting, and disappeared once the rainy season began. Liana-cut trees grew more (0.4 mm/mm year) than liana-uncut trees (0.2 mm/mm year). These findings suggest that lianas may interfere with water availability to these trees during the dry season, and may also hinder tree growth.

Replicative ageing in the fission yeast Schizosaccharomyces pombe.

Saccharomyces cerevisiae has been widely used as a model organism in studies of replicative ageing and senescence. The relevance of these studies to ageing in other organisms has, however, been questioned, since this yeast divides by budding rather than fission, the more common pattern in higher organisms. Here we report that, contrary to popular belief, the fission yeast Schizosaccharomyces pombe also undergoes replicative senescence and in a manner superficially analogous to budding yeast. These experiments provide the first evidence of age asymmetry in cell fission and are consistent with the hypothesis of Jazwinski, that asymmetric division underlies culture immortality. Given their evolutionary divergence, comparison of the ageing determinants in fission and budding yeasts may help identify common mechanisms of the ageing process.

Effect of Cu,Zn superoxide dismutase disruption mutation on replicative senescence in Saccharomyces cerevisiae.

The role of oxidative damage in determining the replicative lifespan of Saccharomyces cerevisiae was investigated using a wild-type haploid laboratory yeast and a Cu,Zn superoxide dismutase (sod1) mutant derivative on glucose, ethanol, glycerol and galactose media. SOD1 expression was necessary to ensure longevity on all carbon sources tested. Whilst carbon source and SOD1 gene expression do influence yeast lifespan, the relationship between the two factors is complex.

Development of a green fluorescent protein reporter for a yeast genotoxicity biosensor.

A reporter system, constructed for a laboratory screen for new genes involved in DNA repair in the brewer's yeast Saccharomyces cerevisiae, has been developed for use in a genotoxicity biosensor. The strain produces green fluorescent protein (yEGFP) when DNA damage has occurred. yEGFP is codon optimised for yeasts. The reporter does not respond to chemicals which delay mitosis, and responds appropriately to the genetic regulation of DNA repair. Data is presented which demonstrate strain improvements appropriate to biosensor technology: improved signal to noise ratio, ease of data collection and uncomplicated material handling.

In vitro metabolism of ceftiofur in bovine tissues.

The metabolism of ceftiofur in bovine kidney, liver, muscle and lung, and the effects of the presence of cystine and glutathione in the media were evaluated using S-9 and microsomal tissue fractions. Conversion of ceftiofur to desfuroylceftiofur (DFC) was catalyzed by an esterase which was most active in kidney, followed by liver. It was not very active in muscle and lung. After DFC was liberated, it rapidly bound primarily to tissue proteins (> 56%), and was also conjugated to cysteine and glutathione. Production of DFC-cysteine by disulfide exchange of DFC with cystine and production of DFC-glutathione by conjugation of DFC to glutathione occurred in buffer if glutathione and cystine were present in the medium. These conjugations were also observed in incubations with tissue fractions, indicating that they were not inhibited by the tissues endogenous molecules. In addition, the metabolism of DFC-glutathione to DFC-cysteine was observed when tissue proteins were present. The metabolism of DFC-glutathione to DFC-cysteine was faster in kidney than in liver. Metabolites devoid of an intact beta-lactam ring were not observed in these in vitro studies.

Gavage administration of the fungal toxin fumonisin B1 to female Sprague-Dawley rats.

The fungal toxin fumonisin B1 (FB1) is a contaminant of corn-based foods and feeds produced by members of the genus Fusarium. Fumonisin B1 toxicity was examined using gavage administration of purified toxin to female Sprague-Dawley rats. For 11 consecutive days each rat received a single dose of FB1 at the following concentrations: control (saline), 1, 5, 15, 35, or 75 mg FB1/kg body weight/d. Significantly depressed body weight and food consumption occurred at 35 and 75 mg FB1/kg/d. By the end of the dosing period there were no significant changes in food consumption. Kidneys and bone marrow were most sensitive to FB1 exposure. Changes in renal morphology were observed from 5 to 75 mg FB1/kg/d, accompanied by transient changes in urine osmolality and urine enzyme levels. Increased cellular vacuolation was the primary change associated with bone-marrow toxicity, starting at doses of 5 mg FB1/kg/d. Hepatotoxicity was indicated by reduced liver weight, elevated serum alanine amonitransferase (ALT), and mild histopathological changes occurring at doses of 15 mg FB1/kg/d and higher. Increased cytoplasmic vacuolation of adrenal cortex cells occurred in rats treated with 15 mg FB1/kg/d and higher, indicating that the adrenals are also potential targets of FB1. Elevated serum cholesterol, which is a consistent response to FB1 was observed at 5 mg FB1/kg/d and higher. Based on responses in this study, gavage is an appropriate substitute for longer feeding studies. Compared to previous work with male rats, gender-related difference in FB1 responses lacked consistency but indicated that males may be marginally more sensitive than female Sprague-Dawley rats.

Synthesis and Structure of the New Ternary Nitride SrTiN(2).

A new ternary nitride, SrTiN(2), has been synthesized by the solid-state reaction of Sr(2)N with TiN and characterized by powder X-ray diffraction. SrTiN(2) crystallizes in the tetragonal space group P4/nmm (a = 3.8799(2) Å, c = 7.6985(4) Å, Z = 2) and is isostructural with KCoO(2). Titanium is coordinated to five nitrogens in a distorted square-based pyramidal geometry, forming layers of edge-sharing pyramids which stack along the (001) direction. Strontium is situated between the Ti-N layers and is coordinated to five nitrogen atoms. The title compound is only the third example of a ternary titanium nitride.

Toxicity of fumonisin B1 to B6C3F1 mice: a 14-day gavage study.

Fumonisin B1 (FB1) is a fungal toxin produced by members of the genus Fusarium. Ingestion of FB1 causes species-specific neurotoxic, nephrotoxic, hepatotoxic and pulmonary effects. The clinical, haematological and pathological responses of adult male and female B6C3F1 mice to FB1 were assessed following 14 daily gavage doses ranging from 1 to 75 mg FB1/kg body weight/day. There were no consistent sex-related changes. Although all responses were modest, the most notable effects of FB1 were on the liver, bone marrow, adrenals and kidneys. In the liver, hepatocellular single cell necrosis, mitosis and anisokaryosis were observed, accompanied by elevated serum ALT. In the kidneys, minor histopathological changes were confined to female mice, while mild decreases in ion transport and increases in blood urea nitrogen were seen only in males. Small changes in glutathione levels were observed in the kidneys and livers of male mice. Adrenal cortical cell vacuolation was observed at 15 mg FB1/kg and higher in females and from 35 mg FB1/kg in males. Serum cholesterol was elevated in both male and female mice, possibly due to FB1-induced changes in lipid metabolism in the liver and adrenals. Although bone marrow cell numbers were unchanged, increases in vacuolated myeloid cells and lymphocytes were observed in female mice. In general, the degree of changes observed indicate that mice are not as sensitive a model of FB1 toxicity as rats.

Ceftiofur hydrochloride: plasma and tissue distribution in swine following intramuscular administration at various doses.

Twelve mixed-breed swine (26.5-42.5 kg) received three daily intramuscular (i.m.) doses of 14C-ceftiofur hydrochloride. Three males and three females, received 6.76 +/- 0.83 mg of 14C-ceftiofur free acid equivalents (CFAE)/kg body weight (b.w.)/day, while the other group received 4.41 +/- 0.97 mg.CFAE/kg b.w./day. The swine were slaughtered 12 h following the last dose. Total dose accountability for the 6.76 mg dose was 91.44 +/- 16.11% (72.51% in urine; 12.63% in faeces). For the 4.41 mg dose, accountability was 100.35 +/- 20.45% (82.48% in urine; 12.85% in faeces). Within the tissues used for residue monitoring, the highest concentrations were observed in the kidneys (10.68 and 6.33 micrograms.CFAE/g for the 6.76 and 4.41 mg doses, respectively), followed by the injection sites, lungs, liver and muscle. In a separate study, twelve mix-breed swine (23.1-39.7 kg) received 14C-ceftiofur hydrochloride at 3.08 mg.CFAE/kg b.w. once daily for 3 days. Two males and two females were slaughtered at either 12, 72 or 120 h after the last dose. Total dose accountability for the 3.08 mg dose was > 83% (> 68% in urine; > 13% in faeces). In swine slaughtered 12 h after last dose, residue concentrations closest to the safe concentrations were observed in the kidneys (3.62 micrograms.CFAE/g), followed by the injection sites, lungs, liver and muscle.

Determination of ceftiofur and its desfuroylceftiofur-related metabolites in swine tissues by high-performance liquid chromatography.

An HPLC method was developed and validated for the determination of ceftiofur-related metabolites that have the potential to be microbiologically active in swine muscle, kidney, liver and fat. Its performance was evaluated against incurred-residue swine tissues. This method is based on the cleavage of the disulfide and/or thioester bonds between the metabolites and their conjugate sulfur containing moiety using dithioerythritol to yield desfuroylceftiofur, and further stabilization to desfuroylceftiofur acetamide. The limit of quantitation was 0.1 micrograms ceftiofur equivalents/g tissue. The assay is specific for ceftiofur-related metabolites when evaluated against commercially available antibiotics for swine.

Preliminary characterization of the structural proteins of the coronaviruses, sialodacryoadenitis virus and Parker's rat coronavirus.

A procedure was developed for the partial purification of the rat coronaviruses, sialodacryoadenitis virus (SDAV) and Parker's rat coronavirus (PRC). The SDAV and PRC were replicated in L-2 cell monolayer cultures, precipitated with ammonium sulphate, and further concentrated using sucrose density gradient centrifugation. The major SDAV and PRC proteins were identified by immunoblotting and compared with those of the JHM strain of mouse hepatitis virus (MHV-JHM). Monoclonal antibodies (MAb) against the M protein of JHM recognized proteins interpreted to be slightly smaller in immunoblots of SDAV and PRC (22.8 vs 23K for JHM). Similarly, a monoclonal antibody against the JHM N protein reacted with proteins of 53K in SDAV and PRC (vs 56 K for JHM). Polyclonal antisera to all three viruses also cross-reacted with the M and N proteins. Some cross-reactivity amongst the S proteins was observed. Based on these data, the structural proteins of the rat coronaviruses, SDAV and PRC are closely related to those of MHV-JHM.

Foliar injury in young Betula pendula roth., Salix purpurea L. and Ilex aquifolium L. trees and in propagated Taxus baccata L. shoots exposed to intermittent fog at a range of acidities.

The occurrence of various visible symptoms of foliar injury was assessed on a 5-point scale for each of the species. There was a distinct increase in the incidence of most types of injury in plant material which has been exposed to pH 2.5 acid fog. No clear pH-related trend in foliar injury occurred in plants exposed to acid fog at pH 3.5, pH 4.5 and pH 5.6, indicating that a threshold for a range of different types of visible injury may exist between pH 2.5 and pH 3.5 for all four species.

'Possession' in psychiatric patients in Britain.

Various different types of explanation that have been offered for so-called possession states are reviewed. Patients continue to be seen who believe that they are possessed. A group of 16 such patients are described. The relative contributions of individual and cultural factors are discussed. It is concluded that in this group of patients, while cultural factors may exert a pathoplastic influence, the main causes of disturbance lie within the individual.

Dyslexia as cause of psychiatric disorder in adults.

A few patients may be of normal intelligence but have difficulties in reading, writing, and spelling, which are the main precipitants of a psychiatric disorder. In seven patients this illiteracy emerged only after intensive examination, as they had hidden it from employers, friends, and children. Characteristically these patients are often very sensitive about this disability and marital friction is common. They are also often noticeably resistant to remedial help.