Mapping synaptic glutamate transporter dysfunction in vivo to regions surrounding Aß plaques by iGluSnFR two-photon imaging.

Amyloid-ß (Aß) plaques, a hallmark of Alzheimer's disease (AD), are surrounded by regions of neuronal and glial hyperactivity. We use in vivo two-photon and wide-field imaging of the glutamate sensor iGluSnFR to determine whether pathological changes in glutamate dynamics in the immediate vicinity of Aß deposits in APPPS1 transgenic mice could alter neuronal activity in this microenvironment. In regions close to Aß plaques chronic states of high spontaneous glutamate fluctuations are observed and the timing of glutamate responses evoked by sensory stimulation exhibit slower decay rates in two cortical brain areas. GLT-1 expression is reduced around Aß plaques and upregulation of GLT-1 expression and activity by ceftriaxone partially restores glutamate dynamics to values in control regions. We conclude that the toxic microenvironment surrounding Aß plaques results, at least partially, from enhanced glutamate levels and that pharmacologically increasing GLT-1 expression and activity may be a new target for early therapeutic intervention.

Genetic analysis of dopaminergic system development in zebrafish.

Zebrafish have become an important model organism to study the genetic control of vertebrate nervous system development. Here, we present an overview on the formation of dopaminergic neuronal groups in zebrafish and compare the positions of DA neurons in fish and mammals using the neuromere model of the vertebrate brain. Based on mutant analysis, we evaluate the role of several signaling pathways in catecholaminergic neuron specification. We further discuss the prospect of identifying novel genes involved in dopaminergic development through forward genetics mutagenesis screens.

Tg.AC genetically altered mouse: assay working group overview of available data.

In a Government/Industry/Academic partnership to evaluate alternative approaches to carcinogenicity testing, 21 pharmaceutical agents representing a variety of chemical and pharmacological classes and possessing known human and or rodent carcinogenic potential were selected for study in several rodent models. The studies from this partnership project, coordinated by the International Life Sciences Institute, provide additional data to better understand the models' limitations and sensitivity in identifying carcinogens. The results of these alternative model studies were reviewed by members of Assay Working Groups (AWG) composed of scientists from government and industry with expertise in toxicology, genetics, statistics, and pathology. The Tg.AC genetically manipulated mouse was one of the models selected for this project based on previous studies indicating that Tg.AC mice seem to respond to topical application of either mutagenic or nonmutagenic carcinogens with papilloma formation at the site of application. This communication describes the results and AWG interpretations of studies conducted on 14 chemicals administered by the topical and oral (gavage and/or diet) routes to Tg.AC genetically manipulated mice. Cyclosporin A, an immunosuppresant human carcinogen, ethinyl estradiol and diethylstilbestrol (human hormone carcinogens) and clofibrate, an hepatocarcinogenic peroxisome proliferator in rodents, were considered clearly positive in the topical studies. In the oral studies, ethinyl estradiol and diethylstilbestrol were negative, cyclosporin was considered equivocal, and results were not available for the clofibrate study. Of the 3 genotoxic human carcinogens (phenacetin, melphalan, and cyclophosphamide), phenacetin was negative by both the topical and oral routes. Melphalan and cyclophosphamide are, respectively, direct and indirect DNA alkylating agents and topical administration of both caused equivocal responses. With the exception of clofibrate, Tg.AC mice did not exhibit tumor responses to the rodent carcinogens that were putative human noncarcinogens, (di(2-ethylhexyl) phthalate, methapyraline HCl, phenobarbital Na, reserpine, sulfamethoxazole or WY-14643, or the nongenotoxic, noncarcinogen, sulfisoxazole) regardless of route of administration. Based on the observed responses in these studies, it was concluded by the AWG that the Tg.AC model was not overly sensitive and possesses utility as an adjunct to the battery of toxicity studies used to establish human carcinogenic risk.

Unique renal tubule changes induced in rats and mice by the peroxisome proliferators 2,4-dichlorophenoxyacetic acid (2,4-D) and WY-14643.

Peroxisome proliferators are non-mutagenic carcinogens in the liver of rodents, acting both as initiators and promoters. The National Toxicology Program (NTP) conducted a study of several peroxisome proliferators (PPs), including Wyeth (WY)-14643 as a prototypical PP and 2,4-dichlorophenoxyacetic acid (2,4-D) as a weak PP, in Sprague-Dawley rats. B6C3F1 mice, and Syrian hamsters. In the kidney, an unusual change was observed in the outer stripe of the outer medulla, especially in rats treated with 2,4-D or WY-14643. This change was characterized by foci of tubules that were partially or completely lined by basophilic epithelial cells with decreased cytoplasm and high nuclear density. Changes typical of chronic nephropathy such as interstitial fibrosis or basement membrane thickening were not associated with these foci. Results of immunohistochemical staining for catalase and cytochrome P-450 4A in the kidney indicated increased staining intensity in renal tubular epithelial cells primarily in the region where the affected tubules were observed: however, the altered cells were negative for both immunohistochemical markers. Ultrastructurally, affected cells had long brush borders typical of the P3 tubule segment. The most distinguishing ultrastructural change was a decreased amount of electronlucent cytoplasm that contained few differentiated organelles and, in particular, a prominent reduced volume and number of mitochondria; changes in peroxisomes were not apparent. In addition to the lesion in rats, mice treated with the highest dose of 2,4-D, but not WY-14643, manifested similar renal tubular changes as seen by light microscopy. Neither chemical induced renal tubular lesions in hamsters. Hepatocellular changes characteristic of PPs were present in all 3 species treated with WY-14643, but not 2,4-D. These results indicate that the rat is the species most sensitive to the nephrotoxic effects of PPs and there is a site specificity to this toxicity related to areas of PP-related enzyme induction. Although 2,4-D is considered a weak PP for the liver, it was the most effective at inducing renal lesions, indicating that the toxic potency of various PPs will depend on the target organ.

A retrospective analysis of background lesions and tissue accountability for male accessory sex organs in Fischer-344 rats.

Because the paired lobes (ventral, dorsal, lateral, and anterior) of the rat prostate have not been consistently sampled in many carcinogenicity and toxicity studies, comparison among different investigations has been compromised. The lack of specific site identification for prostatic lesions further lessens the value of incidences reported. We present here the lobe-specific incidences and degree of severity of background prostatic, seminal vesicular, and ampullary glandular lesions in 1768 control Fischer-344 rats from 35 recent National Toxicology Program 2-year carcinogenicity and toxicity studies conducted in 4 laboratories. The dorsal and lateral lobes were combined and considered the dorsolateral lobe where inflammation, epithelial degeneration, mucinous cysts, and edema were observed. Inflammation in the dorsolateral lobes was significantly associated with pituitary gland adenoma whose prolactin was suggested to play an important role in pathogenesis of prostatic inflammation. Epithelial degeneration, epithelial hyperplasia, inflammation, edema, and adenoma were conspicuous in the ventral lobes. Inflammation and edema occurred in the anterior lobes (coagulating glands). Inflammation, dilatation, epithelial hyperplasia, edema, and adenoma were observed in the seminal vesicles. Inflammation was also present in the ampullary glands. We suggest an optimal embedment and trimming method in rat prostate and seminal vesicle to ensure adequate, consistent sampling.

The design of phenylglycine containing benzamidine carboxamides as potent and selective inhibitors of factor Xa.

Factor Xa, a critical serine protease in the blood coagulation cascade, has become a target for inhibition as a strategy for the invention of novel anti-thrombotic agents. Here we describe the development of phenylglycine containing benzamidine carboxamides as novel, potent and selective inhibitors of factor Xa.

Loss of heterozygosity frequency at the Trp53 locus in p53-deficient (+/-) mouse tumors is carcinogen-and tissue-dependent.

Mutagenic carcinogens rapidly induced tumors in the p53 haploinsufficient mouse. Heterozygous p53-deficient (+/-) mice were exposed to different mutagenic carcinogens to determine whether p53 loss of heterozygosity (LOH) was carcinogen-and tissue-dependent. For 26 weeks, C57BL/6 (N4) [corrected] p53-deficient (+/-) male or female mice were exposed to p-cresidine, benzene or phenolphthalein. Tumors were examined first for loss of the wild-type p53 allele. p-cresidine induced p53 LOH in three of 13 bladder tumors, whereas hepatocellular tumors showed p53 LOH in carcinomas (2/2), but not in adenomas (0/3). Benzene induced p53 LOH in 13 of 16 tumors examined. Finally, phenolphthalein induced p53 LOH in all tumors analyzed (21/21). Analysis of the p-cresidine-induced bladder tumors by cold single-strand conformation polymorphism (SSCP) analysis of exon 4-9 amplicons failed to demonstrate polymorphisms associated with mutations in tumors that retained the p53 wild-type allele. p-cresidine induced a dose-related increase in lacI mutations in bladder DNA. In summary, these data demonstrate that loss of the wild-type allele occurred frequently in thymic lymphomas and sarcomas, but less frequently in carcinomas of the urinary bladder. In the bladder carcinomas other mechanisms may be operational. These might include (i) other mechanisms of p53 inactivation, (ii) inactivating mutations occurring outside exons 4-9 or (iii) p53 haploinsufficiency creating a condition that favors other critical genetic events which drive bladder carcinogenesis, as evidenced by the significant decrease in tumor latency. Understanding the mechanisms of p53 LOH and chemical carcinogenesis in this genetically altered model could lead to better models for prospective identification and understanding of potential human carcinogens and the role of the p53 tumor suppressor gene in different pathways of chemical carcinogenesis.

Inhalation toxicity studies of the alpha,beta-unsaturated ketones: 2-cyclohexene-1-one.

2-Cyclohexene-1-one (CHX) is a cyclic alpha,beta-unsaturated ketone with broad human exposure. CHX is an environmental pollutant and is present in tobacco smoke and in soft drinks sweetened with cyclamate. Interest in the toxicity of this class of compounds is due to their structural similarity to the cytotoxin acrolein. In a pilot study, rats and mice were exposed to 0, 20, 40, or 80 ppm CHX for 6 h/day. The study was terminated after 4 days due to acute toxicity in the high-dose groups. In a subsequent 14-day study, mice and rats were exposed to 0, 2.5, 5, or 10 ppm CHX for 6 h/day. All animals survived exposure until terminal sacrifice. Body weights were not significantly different from controls after 14 days of exposure. Liver/body weights were increased in male and female mice exposed to 5 and 10 ppm, and in male and female rats exposed to 10 ppm CHX. Ninety-day toxicity studies were conducted to provide data required to design chronic toxicity and carcinogenicity studies of CHX if it is determined such studies are necessary. Groups of 10 male and female F-344 rats and B6C3F1 mice were exposed to 0, 2.5, 5, or 10 ppm CHX for 6 h/day for 13 wk. All animals survived until sacrifice. Body weights were not significantly different from controls after 13 wk of exposure. Liver weights were increased in male and female mice exposed to 5 and 10 ppm and in male and female rats exposed to 10 ppm CHX. No adverse effects on bone-marrow micronuclei, sperm motility, or vaginal cytology were observed. Microscopic lesions included hyperplasia, and squamous metaplasia in the nasal cavity in rats and mice of both sexes at all doses. Nasal-cavity erosion and suppurative inflammation also occurred in high-dose mice. Larynx and lung were not affected in either sex or species. Dose-related hepatic centrilobular cytoplasmic vacuolation was seen in male rats only. These data suggest that CHX acts as an alkylating agent primarily producing toxicity at the exposure site.

Consumer, provider, and informal caregiver opinions on psychiatric advance directives.

Psychiatric advance directives (PADs) are legal documents that permit competent adults to make choices in the present about their future psychiatric treatment if they lose their decision-making capacity. PADs may provide for the appointment of surrogate decision-makers. The present project was undertaken to obtain opinions from the consumer (the patient), provider, and informal caregiver/surrogate about the Oregon PAD. Results of this pilot study show that the majority of no-PAD group consumers reported that they did not get enough help with PAD preparation. The PAD group consumers reported feeling empowered by PAD preparation, but at the follow-up interview, they were less enthusiastic and more critical of PAD policy that was relevant to implementation. Many providers also were concerned about successful PAD implementation. PAD legislation alone does not translate into adequate policy.

Mutant models of prolonged life span.

Aging is an important biological process that affects all creatures. For humans, age-related diseases and the question of why we age and die also have tremendous social and philosophical impact. We can therefore expect that models to study mechanisms of the aging process will always attract much interest. Until recently, the mutant model approach to study molecular mechanisms of aging has been limited to lower animals such as yeast, worms, and flies. However, given the current power of genetic technology in mammals, we can expect that phenotypes of prolonged life span will increasingly be seen in mice and subject to evaluation by pathologists. A brief review of current models is presented.

Induction of lung lesions in female rats following chronic exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin.

2,3,7,8,-Tetrachlorodibenzo-p-dioxin (TCDD) has been classified as a known human carcinogen, and epidemiologic studies identify the lung as one of the target organs. Few experimental studies have attempted to characterize pulmonary effects of TCDD exposure. In this study, we characterize the induction of lesions in the lung by chronic oral TCDD exposure in diethylnitrosamine (DEN)-initiated or noninitiated female Sprague-Dawley rats. Two or 18 weeks after initiation, rats were treated with TCDD continuously for 14, 30, or 60 weeks by biweekly oral gavage (1,750 ng TCDD/kg) at a dose equivalent to 125 ng/kg body weight per day (controls received corn oil). To assess the time dependence and reversibility of potential changes, some groups included withdrawal periods of 16 or 30 weeks after 30 weeks of TCDD treatment. TCDD treatment alone for 60 weeks caused significant increases in alveolar-bronchiolar (AB) metaplasia. TCDD treatment of DEN-initiated animals for 60 weeks resulted in a significant increase in bronchiolar epithelial hyperplasia. These increases were not observed in animals treated with TCDD for 30 weeks followed by corn oil for 30 weeks, indicating that the development of these lesions required continuous exposure to TCDD. AB hyperplasia increased in an age-dependent manner after DEN initiation but was unaffected by TCDD treatment. Expression of the aromatic hydrocarbon receptor (AHR) and induction of CYP1A1 was observed only in bronchiolar Clara and ciliated cells, indicating that the mechanism of induction of AB metaplasia may be mediated by the AHR. TCDD elimination half-life was monophasic in the lung, and serum and was estimated to be 39.7 days and 44.6 days, respectively, independent of age, tissue TCDD concentration, or body weight. This is the first report to identify the AB region as a target for TCDD-induced metaplastic and proliferative changes after chronic oral exposure.

Genetic disruption of Ptgs-1, as well as Ptgs-2, reduces intestinal tumorigenesis in Min mice.

Two isoforms of cyclooxygenase (COX) are known, and to date most studies have implicated COX-2, rather than COX-1, as the isoform involved in colon carcinogenesis. In the present study, we show that homologous disruption of either Ptgs-1 or Ptgs-2 (genes coding for COX-1 or COX-2, respectively) reduced polyp formation in Min/+ mice by approximately 80%. Only COX-1 protein was immunohistochemically detected in normal intestinal tissue, whereas both COX-1 and variable levels of COX-2 protein were detected in polyps. Prostaglandin E2 was increased in polyps compared with normal tissue, and both COX-1 and COX-2 contributed to the PGE2 produced. The results indicate that COX-1, as well as COX-2, plays a key role in intestinal tumorigenesis and that COX-1 may also be a chemotherapeutic target for nonsteroidal anti-inflammatory drugs.

A farnesyl transferase inhibitor suppresses TPA-mediated skin tumor development without altering hyperplasia in the ras transgenic Tg.AC mouse.

The Tg.AC mouse carries an activated v-Ha-ras oncogene fused to an embryonic zeta-globin promoter and develops cutaneous papillomas in response to specific chemicals, full thickness wounding, and ultraviolet radiation. Papilloma development in these mice has been suggested to be dependent upon activation of ras transgene expression, thus providing a potential model for studying ras-inhibitory compounds. Farnesyl transferase inhibitors (FTIs) prevent a critical posttranslational modification step necessary for activation of ras proteins. Our studies demonstrated that a tricyclic FTI (SCH 56582) applied directly to the skin of homozygous Tg.AC mice 1 h prior to administration of the tumor promoter TPA decreased tumor multiplicity compared to TPA-only controls. In addition, a reduction of TPA-induced tumor development was seen in similarly treated hemizygous Tg.AC mice either on an FVB/N strain background or 50% C57BL/6. Histological examination of skin from Tg. AC(+/-):FVB/N mice revealed no differences with respect to 12-O-tetradecamoylpharbol-13-acetate (TPA)-mediated hyperplasia. Keratinocytes isolated from treated and control skin were assayed for ras transgene expression by reverse transcription-polymerase chain reaction, and expression was detected in both TPA- and FTI+TPA-treated tissue, although the appearance of transgene positive pre-papillomas was observed only in histological sections taken 21 d after the first treatment. In summary, we have used a regimen of topical application of an FTI (SCH 56582) to suppress TPA-mediated papillomagenesis in v-Ha-ras transgenic Tg.AC mice. These studies demonstrate that TPA-induced epidermal hyperplasia is a ras-independent process, while papilloma development in response to TPA treatment requires the function of activated ras.

Extragonadal teratocarcinoma in chimeric mice.

Chimeric mice often are created through the genetic manipulation of the mouse embryo in the process of developing animal models of disease. These mice have variable percentages of their somatic and germ cells derived from the donor embryonic stem cells and host blastocysts. In the development of mouse models deficient in the breast cancer susceptibility gene 2 (Brca2) or the 70-kd heat shock protein (Hsp70-2), 3-4-week-old chimeras developed single or multiple masses composed of both well-differentiated and poorly differentiated tissues derived from all three germ layers. These cases of extragonadal teratocarcinoma, a rarely reported tumor, may be related to the genetic predisposition of the 129/Ola mouse strain used to generate the embryonic stem cells.

New issues and future legislation on biosafety.

The current debate on gene technology in Europe is reviewed with particular emphasis on the role of EFB and science in general. From this debate important lessons have been learned by scientists, industrial companies, legislators, parliamentarians and interest organisations. This gives confidence that the continued debate will eventually lead to public acceptance of gene technology even in the food area.

Safe biotechnology 9: values in risk assessment for the environmental application of microorganisms. The Safety in Biotechnology Working Party of the European Federation of Biotechnology.

Risk assessment for the deliberate release of microorganisms into the environment is traditionally carried out on a case-by-case basis. In a similar approach to that used when assessing human pathogenicity, we propose an alternative approach by introducing risk classes to facilitate or complement this type of risk assessment. These consider several sets of scenarios that address the different values that need to be protected. Examples of this approach include risk-class definitions for soil fertility and biodiversity.