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J Ashby

Publications and source records attributed to J Ashby.

At least 19 recordsLinked to original sources
PubMed1995

Enhanced hepatocyte colony growth in soft agar after in vivo treatment with a genotoxic carcinogen: a potential assay for hepatocarcinogens?

We have shown previously that approximately 1 in 10,000 primary hepatocytes isolated from untreated rats undergo clonal growth in soft agar in vitro in response to the synergistic action of nafenopin, a peroxisome proliferator (PP) and epidermal growth factor (EGF), a naturally occurring liver growth regulator. Here, we demonstrate that prior treatment of the animals with the genotoxic hepatocarcinogen diethylnitrosamine (DEN) caused a dose-dependent increase in soft agar colony numbers formed in vitro. These data suggest that the colony assay may offer a method of detecting in vitro hepatocytes transformed in vivo by DEN. It is known that rats treated with DEN develop enzyme altered foci prior to the development of tumours. The majority of these foci express high levels of gamma-glutamyl transpeptidase (GGT). However, foci promoted by PPs do not show this increased enzyme activity. In the present study, the colonies we have generated in vitro mimicked this pattern since the majority (approximately 80%) of the spontaneous colonies expressed GGT whereas colonies promoted by the synergistic action of nafenopin and EGF were mainly (75%) GGT negative. The proportion of colonies positive for GGT were similar using either hepatocytes isolated from control or from DEN-initiated rats. Further studies are required to assess if the hepatocytes selected for clonal expansion by this EGF/nafenopin regime reflect the presumed pre-neoplastic cells induced by genotoxin in vivo and associated with an increased propensity to cancer.

Agar↗
PubMed1995

Consideration of the liver of embryonic lacZ transgenic mice as an analogue of the mouse coat colour spot test: preliminary data and technical problems.

Pregnant lacZ+ transgenic mice (Muta Mouse) were treated with ENU (25 mg/kg) by oral gavage on day 10.5 of pregnancy. This dose of ENU is the optimal dose observed by other investigators for activity in the mouse coat colour spot test. Day 10.5 of pregnancy represents the stage when the embryonic liver first becomes visually discernable. By day 15.5, when the maternal and embryonic livers were analysed for lacZ- mutation frequency, the embryonic liver is the largest tissue in the embryo (approximately 25 mg). These experiments therefore represent treatment of a small pool of progenitor hepatocytes just as they are entering into an intense wave of cell division--optimum conditions for the fixation of mutations. Exposure to ENU led to an average fourfold, and a maximum tenfold increase in mutation frequency in the embryonic livers. This relatively weak response is consistent with lacZ- mutants not having a growth advantage; unlike in the mouse spot test, clonal amplification of lacZ- mutants cannot be separately scored. The level of mutation in the control embryonic livers was lower than that of the maternal control livers, but the group sizes were too small to conclude this definitively. Embryonic livers from each individual mother showed a range of mutation frequencies that were not obviously related to that of the maternal liver. On a treatment group basis, ENU was non-mutagenic to maternal livers. Half of the embryonic livers yielded DNA that failed to package, despite repeated attempts and re-isolation of the DNA from the liver. The cause of this unexpected finding is not clear. What is clear is that it was not due to the ENU treatment because eight of 22 control embryonic livers behaved similarly. These preliminary results suggest that further research is required in order to establish a practical transgenic analogue of the mouse coat colour spot test.

Animals↗
PubMed1995

The rodent dominant lethal assay: a proposed format for data presentation that alerts to pseudo-dominant lethal effects.

The rodent dominant lethal (DL) germ cell mutagenicity assay is the primary test for possible human germ cell mutagens. As such, it occupies a critical regulatory position. DL assay data are often difficult to assess because of the quantity of data involved, and because several related assay parameters require to be considered simultaneously. To reduce this difficulty a schematic method of data presentation is proposed and illustrated. This method enables the most pertinent assay data and parameters to be viewed and considered simultaneously. Using this format of data presentation, existing DL studies on cyclophosphamide, methylnitrosourea, diethylhexylphthalate, divinyl sulphone, methyl methanesulphonate, 6-mercaptopurine and ethylenethiourea are re-analysed.

Animals↗
PubMed1995

Chemicals for evaluating the sensitivity and specificity of reduced/transgenic rodent cancer bioassay protocols.

The standard two-species/two-gender lifetime carcinogenicity bioassay protocol is not practical when assessing the possible carcinogenicity of a large number of chemicals. This has led to consideration of reduced cancer bioassay protocols (e.g., male rat/female mouse), or to the use of genetically altered rodents that succumb to chemically induced cancer in a fraction of their lifespans. Two uncertainties accompany these endeavours. First, that use of reduced protocols may lead to the non-detection of some weak or tissue-specific carcinogens. Second, that genetically altered rodents may be so sensitive to chemical disturbance of homoeostasis that their carcinogen specificity may be low. Such uncertainties lead to the requirement that the modified test protocols should be validated using appropriate carcinogens and non-carcinogens. Given that several such studies may take place over the coming years it seems appropriate that a common pool of chemicals should be agreed upon for this purpose. To this end a selection of human carcinogens, and rodent carcinogens/non-carcinogens defined by the US National Toxicology Program (NTP), have been collected together. Six categories of chemicals are listed: (1) representative human carcinogens; (2) representative trans-species genotoxic carcinogens; (3) all two-species non-carcinogens reported to be negative in the Salmonella mutation assay and lacking structural alerts to electrophilicity; (4) representative single-species/presumed non-genotoxic carcinogens; (5) all two-species non-carcinogens that are mutagens in the Salmonella mutation assay and which are structurally alerting; (6) all chemicals possessing equivocal evidence of carcinogenicity and which are both structurally alerting and mutagenic to Salmonella. Chemicals in the first four groups provide robust calibrants for determining the sensitivity and specificity of reduced/accelerated rodent carcinogenicity bioassay protocols. Chemicals in the last two groups may be of value when studying the interface between the sensitivity and the specificity of modified rodent carcinogenicity bioassay protocols.

Animals↗
PubMed1995

Comparison of lacI and lacZ transgenic mouse mutation assays: an EU-sponsored interlaboratory study.

As part of an EU Environment Programme sponsored study, the performance of two transgenic mouse mutation assays has been evaluated in three laboratories using common liver samples. The systems studied were the lacI-(Big Blue) mutation assay, and the GalE- positive selection modified lacZ- (Muta Mouse) assay. The liver samples compared were derived from mice treated with either saline or dimethylnitrosamine (DMN; 14 day recovery). Each assay gave an increased mutation frequency (MF) for the DMN-treated livers when compared to the saline control MFs. Sources of variability in the assays are discussed, and it is concluded that whole liver should be homogenised before DNA extraction, and that concurrent controls should be processed in parallel with test samples.

Animals↗
PubMed1995

Effect of essential fatty acids on natural cytotoxicity in patients with colorectal cancer.

Essential fatty acids (EFAs), have been shown to modulate lymphocyte reactivity and destroy various tumour cells in vitro. Natural cytotoxicity, mediated by NK and LAK cells, is believed to play an important anti-cancer role in vivo. The effect of EFAs, given orally as dietary supplementation, on NK and LAK cell cytotoxic activity in patients with localized (n = 10) and advanced (n = 20) colorectal cancer has been studied, using in-vitro 51Cr release cytotoxicity assays with K562 (NK) and DAUDI (LAK) cells. The activity of effector cells was expressed as lytic units. NK cell activity showed no significant change following 15 days ingestion of EFAs in the group with localized cancer, but was significantly reduced in the group with advanced disease and continued to decline, reaching minimal levels following 6 months supplementation (P = 0.017). LAK cell activity showed no overall alteration after 15 days ingestion of EFAs in patients with localized cancer, but in the group with advanced disease, the reduction in the activity occurred at day 15 and steadily declined on prolonged intake, reaching significant minimal levels after 6 months of supplementation (P = 0.05). Cell surface marker analysis (FACS-CD MABs) revealed reduced absolute numbers of CD16+, CD56+ and CD57+ lymphocytes (P < 0.05) in the patients with advanced colorectal cancer. More importantly, the cytotoxicity of NK and LAK cells returned to the pre-supplementation values, 3 months after cessation of EFA intake. Furthermore, there was no alteration in the cytotoxic activity of NK and LAK cells in the control group (advanced colorectal cancer without EFA supplementation) during the 6 months period of evaluation. These results suggest that prolonged EFA supplementation, in the doses used in this study, may have detrimental effects on natural anti-cancer cytotoxic mechanisms in patients with malignant disease.

Aged↗
PubMed1995

High resolution rodent bone marrow micronucleus assays of 1,2-dimethylhydrazine: implication of systemic toxicity and individual responders.

1,2-Dimethylhydrazine (DMH) is confirmed as active in male CBA mouse bone marrow micronucleus (MN) assays conducted at non-toxic dose-levels between 25 and 45 mg/kg. Previous reports of the activity of DMH in rodent bone marrow MN assays are reviewed. By the design of the present experiments it has been possible to confirm the advice offered by the United Kingdom Environmental Mutagen Society (UKEMS) that once 2000 polychromatic erythrocytes have been assessed per animal, the use of larger animal group sizes becomes the critical means to increase assay sensitivity. Also, the use of historical control data is shown to enhance assay sensitivity at low dose-levels by enabling recognition of individual animals responding to the test chemical. These outliers are seen before the group mean MPE value has been increased enough to attain group statistical significance. It is concluded that careful consideration of test data, within the context of concurrent and historical control data, is sufficient to discern activity in the assay.

1,2-Dimethylhydrazine↗
PubMed1995

The single cell gel electrophoresis assay for induced DNA damage (comet assay): measurement of tail length and moment.

Cultured hepatocytes have been treated with either DMSO or dimethylnitrosamine (NDMA) for either 1 or 2 h and the cells assessed for DNA-damage using the single cell alkaline gel electrophoresis assay (comet assay). A strong positive test response was observed producing comet tails of a length and DNA content not observed in either viable or dead control cells. A stronger test response was observed after a 2 h, as opposed to a 1 h, incubation of hepatocytes with NDMA. The method of processing the image of the comet is discussed and it is proposed that measurement of the length of the comet tail should commence at the estimated trailing edge of the cell, rather than at the leading edge or the estimated centre of the cell. Using this criterion, many control cells have no comet trails thereby enabling chemically induced tails to be more readily assessed. A simplified version of defining the comet tail moment is proposed.

Animals↗