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Gas clouds in present-day galaxies are inefficient at forming stars. Low star-formation efficiency is a critical parameter in galaxy evolution: it is why stars are still forming nearly 14 billion years after the Big Bang and why star clusters generally do not survive their births, instead dispersing to form galactic disks or bulges. Yet the existence of ancient massive bound star clusters (globular clusters) in the Milky Way suggests that efficiencies were higher when they formed ten billion years ago. A local dwarf galaxy, NGC 5253, has a young star cluster that provides an example of highly efficient star formation. Here we report the detection of the J = 3→2 rotational transition of CO at the location of the massive cluster. The gas cloud is hot, dense, quiescent and extremely dusty. Its gas-to-dust ratio is lower than the Galactic value, which we attribute to dust enrichment by the embedded star cluster. Its star-formation efficiency exceeds 50 per cent, tenfold that of clouds in the Milky Way. We suggest that high efficiency results from the force-feeding of star formation by a streamer of gas falling into the galaxy.

Assessment of the safety of nutrients presents a challenge different from that posed by the assessment of other chemicals in food such as additives or contaminants. Because nutrients are essential, a dose-response relation exists at both ends of the intake range, separated by a safe range of intake that reflects normal homeostatic processes. The safe intake may not be the same for all population groups and life stages. The size of the safe intake range for each nutrient will vary and in a few cases may be very small. Certain nutrients such as vitamin A and manganese have known and potentially serious adverse effects at high intakes, whereas others such as iron or vitamin C may have more minor adverse effects that are readily reversible and may only be associated with supplement intake. The risk of harm occurring from taking dietary supplements will depend on the safe intake range of the nutrient concerned, the susceptibility of the individual, and the likely intake of the same nutrient from other supplements or the rest of the diet. In many cases, the available database for the safety of nutrients is very limited because the studies, where available, were not designed to assess adverse effects but may have detected problems when they occurred. Further information on the safety of nutrients could be obtained through careful experimental design. Copyright © 2007 American Society for Nutrition [ABSTRACT FROM AUTHOR]

This is an introduction to a US government program that conducted high-contrast imaging experiments with an electron multiplying charge coupled device (EMCCD) in an interferometric coronagraph. This report will introduce the concepts of "charge blooming" and "starlight saturation" in the context of high-contrast astronomical imaging. These phenomena adversely effect the performance of high-contrast photon-counting instruments that do not use a mask to physically block starlight in the science channel of the coronagraph. The problems will be presented with the help of images taken with a commercial EMCCD camera in the visible nulling coronagraph at the Goddard Space Flight Center. A new clocking scheme for EMCCDs-variable multiplication gain clocking-will be proposed as a means for suppressing horizontal blooming and starlight saturation in an astronomical camera. This opening report will conclude with a discussion of design considerations for a new controller for high-contrast photon-counting with an EMCCD in a coronagraphic instrument. This controller will allow a single frame from an EMCCD to be scanned in multiple modes-photon-counting and digitization-with a variable multiplication gain clock to enable direct imaging of an exoplanet and wavefront control of a coronagraph, simultaneously.

Genotoxic substances are generally not permitted for deliberate use in food production. However, an appreciable number of known or suspected genotoxic substances occur unavoidably in food, e.g. from natural occurrence, environmental contamination and generation during cooking and processing. Over the past decade a margin of exposure (MOE) approach has increasingly been used in assessing the exposure to substances in food that are genotoxic and carcinogenic. The MOE is defined as a reference point on the dose-response curve (e.g. a benchmark dose lower confidences limit derived from a rodent carcinogenicity study) divided by the estimated human intake. A small MOE indicates a higher concern than a very large MOE. Whilst the MOE cannot be directly equated to risk, it supports prioritisation of substances for further research or for possible regulatory action, and provides a basis for communicating to the public. So far, the MOE approach has been confined to substances for which carcinogenicity data are available. In the absence of carcinogenicity data, evidence of genotoxicity is used only in hazard identification. The challenge to the genetic toxicology community is to develop approaches for characterising risk to human health based on data from genotoxicity studies. In order to achieve wide acceptance, it would be important to further address the issues that have been discussed in the context of dose-response modelling of carcinogenicity data in order to assign levels of concern to particular MOE values, and also whether it is possible to make generic conclusions on how potency in genotoxicity assays relates to carcinogenic potency., (© Crown copyright 2015.)

Massive present-day early-type (elliptical and lenticular) galaxies probably gained the bulk of their stellar mass and heavy elements through intense, dust-enshrouded starbursts--that is, increased rates of star formation--in the most massive dark-matter haloes at early epochs. However, it remains unknown how soon after the Big Bang massive starburst progenitors exist. The measured redshift (z) distribution of dusty, massive starbursts has long been suspected to be biased low in z owing to selection effects, as confirmed by recent findings of systems with redshifts as high as ~5 (refs 2-4). Here we report the identification of a massive starburst galaxy at z = 6.34 through a submillimetre colour-selection technique. We unambiguously determined the redshift from a suite of molecular and atomic fine-structure cooling lines. These measurements reveal a hundred billion solar masses of highly excited, chemically evolved interstellar medium in this galaxy, which constitutes at least 40 per cent of the baryonic mass. A 'maximum starburst' converts the gas into stars at a rate more than 2,000 times that of the Milky Way, a rate among the highest observed at any epoch. Despite the overall downturn in cosmic star formation towards the highest redshifts, it seems that environments mature enough to form the most massive, intense starbursts existed at least as early as 880 million years after the Big Bang.

Dust is found throughout the universe and plays an important role for a wide range of astrophysical phenomena. In recent years, new IR facilities have provided powerful new data for understanding these phenomena. However, interpretation of these data is often complicated by a lack of complementary information about the optical properties of astronomically relevant materials. The Optical Properties of Astronomical Silicates with Infrared Techniques (OPASI-T) program at NASA's Goddard Space Flight Center is designed to provide new high-quality laboratory data from which we can derive the optical properties of astrophysical dust analogues. This program makes use of multiple instruments, including new equipment designed and built specifically for this purpose. The suite of instruments allows us to derive optical properties over a wide wavelength range, from the near-IR through the millimeter, also providing the capability for exploring how these properties depend upon the temperature of the sample. In this paper, we discuss the overall structure of the research program, describe the new instruments that have been developed to meet the science goals, and demonstrate the efficacy of these tools., (© 2011 Optical Society of America)

Risk assessment of chemicals in food is generally based upon the results of toxicological studies in laboratory animals, allowing for uncertainties relating to interspecies differences, human variability, and gaps in the database. Use of quantitative human data is preferable if available, as in the example of methylmercury. Methylmercury is a neurotoxic environmental contaminant, for which fish is the main source of dietary exposure. Human data from poisoning incidents and epidemiological studies have been used by expert committees to derive a guideline intake level for methylmercury, based on the susceptibility of the most sensitive lifestage, the developing fetus. In the UK, an expert group of nutritionists and toxicologists was formed to review the benefits and risks associated with fish consumption. A formal risk-benefit analysis was not possible because the nutritional data were not sufficiently quantitative. The Committee on Toxicity of Chemicals in Food, Consumer Products and the Environment (COT), therefore, modified the risk assessment approach to derive different guideline intake levels for different subgroups of the population. The COT opinion was used to provide targeted advice on how much fish can be consumed without undue risk from the contaminants. Consumption by adults of one weekly portion (140 g) of shark, swordfish or marlin, would lead to an exceedance of the guideline intake for methylmercury of 40-90%, set to protect the developing fetus, without considering intake from the rest of the diet. Pregnant women and women who may become pregnant within 1 year were, therefore, advised to avoid consumption of these species. Intakes in other adults would be within a higher guideline intake, set to protect groups of the population other than the developing fetus. However, consumption by children of one weekly portion of these species could lead to an exceedance of this guideline intake by up to 60%, without considering intake from the rest of the diet. It was, therefore, advised that consumption of these species by children should be avoided.

The results of measurements of the refractive index and power attenuation coefficient of Zitex at 290, 77, and 4 K in the spectral region from 1 to 1000 microm are presented. Zitex is a porous Teflon sheet with a filling factor of approximately 50% and is manufactured in several varieties as a filter paper. Zitex is found to be an effective IR block, with thin (200-microm) sheets transmitting less than 1% in the 1-50-microm range while attenuating < or = 10% at wavelengths longer than 200 microm. Some variation in the cutoff wavelength is seen, tending to be a shorter-wavelength cutoff for a smaller pore size. In addition, the thermal conductivity of Zitex at cryogenic temperatures has been measured and is found to be roughly one half that of bulk Teflon. Finally, its dielectric constant has been measured in the submillimeter as n = 1.20, resulting in extremely low dielectric reflection losses. As a result, Zitex is particularly useful as an IR blocking filter in low-noise heterodyne receivers; in the millimeter-wave range (lambda > or = 850 microm or nu < or = 350 GHz) the attenuation of alpha < or = 0.01 cm(-1) for a 3.5-mm thickness filter of Zitex G125 would raise receiver noise temperatures by <1 K.

We present the design of a harmonic resonant filter that can be used with a Fourier transform spectrometer (FTS) for simultaneous measurement of a series of lines in the CO rotational ladder. To enable studies of both broad CO absorptions in Venus and modestly red-shifted CO emission from external galaxies, relatively broad (approximately 10-30-GHz FWHM) transmission passbands are desirable. Because a single low-finesse Fabry-Perot (FP) etalon has insufficient interline rejection, a dual-FP etalon was considered. Such a design provides significantly better interband rejection and somewhat more flattopped transmission spikes. A prototype filter of this type, made of two thin silicon disks spaced by an air gap, has been constructed and used with our FTS at the Caltech Submillimeter Observatory for simultaneous measurement of the four submillimeter CO transitions in the atmosphere of Venus that are accessible from the ground.

To evaluate risk from dermal exposure, the amount of material on the skin must first be measured. The potential for dermal uptake must then be assessed for the potential health effects from systemic exposure. No standard methods exist for studying these processes, and published data are not comparable because of the different techniques used. Future validated methodology should provide a sound scientific basis for risk assessment. Methods for measuring skin and surface contamination will require development of reference contaminated surfaces and skin as part of quality control procedures. Biological monitoring is a valuable tool in the assessment of dermal absorption, in contributing to the validation of in vitro techniques, and in risk assessment and management. It will be necessary to conduct detailed investigations to support risk assessment for dermal exposure. Ultimately, predictive models will be established for exposure and for dermal absorption to support a generic approach and allow risk assessment strategies appropriate to actual workplace situations.

The relationship between food and cancer is extremely complex. It is generally accepted that diet is a contributory factor in the aetiology of a large proportion of cancers, but with very few exceptions, we are unable to identify specific causal agents. Many food components have genotoxic potential and more are produced endogenously during digestion. Conversely, there is increasing evidence that consumption of some foods may decrease the risk of cancer, and a number of plant constituents have been shown to have the potential to inhibit various stages of the carcinogenic process. Yet we have little understanding of the interactions between the different food-related genotoxic and protective factors. A further complication is the variation in individual susceptibility and vulnerability. As a result we are still not able to determine the optimal diet for minimising cancer risk. In recognition of these issues, the UK Ministry of Agriculture, Fisheries and Food (MAFF) is funding a number of projects aimed at providing greater mechanistic understanding of the links between food and cancer, in order to offer detailed advice to the public. This report summarises the proceedings of a workshop entitled 'Factors influencing the carcinogenicity of food chemicals', held in London on 1 June 1998, providing overviews of some of the key issues, and demonstrating how the MAFF-funded research is contributing to advances in these areas. It includes discussion of genetic polymorphisms and how they may contribute to individual susceptibility and help to identify causal links between food components and colorectal cancer. Biomarkers of DNA damage in human studies and of inhibition of carcinogen activation and endogenous formation of genotoxic reactive nitrogen species are examined. Also considered are the potential uses of physiologically based pharmacokinetic modelling techniques for providing more accurate estimates of risk and reducing the uncertainty in extrapolation between species and doses. Research now in progress will help to establish the critical risk and protective factors involved in diet-related colorectal cancers, in order to provide a sound scientific basis for formulation of dietary advice to the public.

Damage to the skin induced by chemical irritants is associated with the release of arachidonic acid metabolites, such as prostaglandin E(2) (PGE(2)) which plays an important role in epidermal inflammation. This study investigated cytotoxicity and release of PGE(2) in human epidermal keratinocytes following an 18 hr exposure of confluent cultures to various skin irritants. The concentration-dependent release of PGE, into the extracellular medium appeared to fall into two categories, which was reflective of possible mechanisms of action. Potent skin irritants, such as phorbol-12-myristate-13-acetate, benzalkonium chloride and tributyltin chloride, elicited an increase in extracellular PGE(2) levels at concentrations that did not produce overt cell damage (uptake of neutral red at these concentrations was comparable to control levels). Non-irritants (2-methoxyethanol and 2-butoxyethyl acetate) and two less severe irritants (sodium dodecyl sulfate and acetic acid) stimulated release of PGE(2) only at concentrations that compromised cellular integrity (uptake of neutral red was at least 50% lower than that of control cultures).

We constructed a 24-pixel bolometer camera operating in the 350- and 450-μm atmospheric windows for the Caltech Submillimeter Observatory (CSO). This instrument uses a monolithic silicon bolometer array that is cooled to approximately 300 mK by a single-shot (3)He refrigerator. First-stage amplification is provided by field-effect transistors at approximately 130 K. The sky is imaged onto the bolometer array by means of several mirrors outside the Dewar and a cold off-axis elliptical mirror inside the cryostat. The beam is defined by cold aperture and field stops, which eliminates the need for any condensing horns. We describe the instrument, present measurements of the physical properties of the bolometer array, describe the performance of the electronics and the data-acquisition system, and demonstrate the sensitivity of the instrument operating at the observatory. Approximate detector noise at 350 μm is 5 × 10(-15) W/√Hz, referenced to the entrance of the Dewar, and the CSO system noise-equivalent flux density is approximately 4 Jy/√Hz. These values are within a factor of 2.5 of the background limit.

An in vitro cell culture approach was evaluated for its ability to provide data pertinent to the assessment of skin irritation potential. The hypothesis of this approach is that a direct toxic insult to the epidermal keratinocyte in vivo may lead to release of inflammatory mediators, which are responsible for initiation of a local primary skin irritant reaction. This paper presents data on the cytotoxic potential of a number of structurally unrelated chemicals (chloroform, 2-methoxyethanol, 2-butoxyethylacetate, toluene, 1-butanol, acetaldehyde, n-hexane, sodium dodecyl sulfate, benzalkonium chloride, silver nitrate, dibutyltin dichloride and tributyltin chloride). Cytotoxicity (neutral red uptake and intracellular acid phosphatase activity) of a number of structurally unrelated chemicals, representative of a wide range of skin irritation potential, was evaluated in cultures of rat and human epidermal keratinocytes. The sensitivity of human and rat keratinocytes to the test chemicals was very similar, irrespective of the endpoint of cytotoxicity. The neutral red uptake assay appeared more generally applicable to the diverse range of chemical structures represented in this study, since not all test chemicals elicited an early increase in intracellular acid phosphatase activity. The results were very encouraging, as a good correlation was evident between cytotoxicity in rat keratinocytes and the degree of erythema and oedema associated with an in vivo skin irritant response in rabbits. Keratinocyte cytotoxicity data may provide an indication of the potential of a chemical to induce a severe skin irritant reaction, or if a chemical is more likely to be a marginal or non-irritant. However, the data illustrate that such assays appear unable to discriminate correctly between more subtle classes of irritancy, such as non-irritant, mild, moderate or severe. Available human in vivo skin irritation data were insufficient to conclude which cell type is preferable for evaluation of human skin irritation potential.

The interaction of tumour promoters with the target cell type (keratinocyte) may be an essential feature of their promoting activity and their ability to initiate an inflammatory response. The role of prostaglandin E(2) (PGE(2)), particularly in the keratinocyte, remains largely unknown, but it is closely associated with inflammation and regenerative epidermal hyperplasia, which appear critical for tumour promotion. Rat keratinocytes derived from sublingual mucosa represent a suitable model to investigate the ability of several irritants, of varying tumour-promoting potency, to stimulate PGE(2) release. Cytotoxicity was evaluated by the neutral red uptake assay and a concentration that reduced cell viability to 50% of control was selected as a maximum concentration for subsequent measurement of PGE(2) release. Phorbol-12-myristate-13-acetate, ionophore A23187 and mezerein stimulated PGE(2) release at non-toxic concentrations. Anthralin, benzoyl peroxide, sodium dodecyl sulfate and acetic acid did not stimulate PGE(2) at non-toxic concentrations, but release was associated with a toxic response. Epidermal growth factor and phospholipase C, which are closely associated with intracellular signalling systems that modulate keratinocyte proliferation and differentiation, also stimulated PGE(2) release. Epidermal growth factor elicited PGE(2) release at a concentration reported to be mitogenic in keratinocyte cultures. The stimulation of PGE(2) release in the absence of a toxic response by PMA, mezerein and ionophore A23187 may be indicative of a direct interaction of the chemicals with intracellular pathways involved in regulation of keratinocyte differentiation and proliferation. This interaction may also reflect the ability of such chemicals to initiate an inflammatory response. Measurement of PGE(2) release may be useful to investigate further the mechanism of action of tumour promoters in the target cell type. In contrast, other tumour promoters did not stimulate release at non-toxic concentrations, which implies that their ability to initiate an inflammatory response and possibly their promoting activity is associated with the induction of a toxic response in the target cell population.

Skin irritation, inflammation and hyperplasia appear to be intimately associated with the phenomenon of tumour promotion, but the mechanism of action remains elusive. Prostaglandins and leukotrienes play an important role in skin inflammation and prostaglandin E(2) (PGE(2)) modulates several events associated with phorbol ester-induced tumour promotion. This study investigated the release of eicosanoids (PGE(2) and leukotriene B(4)) and markers of cytotoxicity [neutral red (NR) uptake and intracellular acid phosphatase (AP) activity], after exposure of rat tongue epithelial (RTE) keratinocyte cultures to chemicals of different irritating and tumour promoting activity. The potent phorbol ester tumour promoter phorbol-12-myristate-13-acetate (PMA), and the less potent, structurally related diterpene ester, mezerein (MEZ) were compared with the known skin irritant sodium dodecyl sulfate (SDS). Cytotoxicity data reflected the in vivo skin irritation potential of the test chemicals and intracellular AP activity was increased after exposure to SDS (160 mug/ml), but did not appear to be increased by the more cytotoxic chemicals PMA and MEZ. Extracellular levels of PGE(2) were increased (200 to > 1000% of control levels) after an 18-hr exposure to PMA or MEZ over a concentration range of 0.01 to 20 mug/ml (NR(50) values 8.0 +/- 6.6 and 15.5 +/- 4.8 mug/ml, respectively). These data indicated that PGE(2) release occurred in the absence of cytotoxicity. In contrast, SDS only elicited PGE(2) release after exposure to 80 mug/ml (a cytotoxic dose level, NR(50) 82.5 +/- 9.9 mug/ml). The potency of a chemical to elicit PGE(2) release in keratinocytes in the absence of a cytotoxic response may reflect intracellular pathways intimately associated with the initiation of an inflammatory response and possibly with tumour promoting activity.

Prostaglandin E2 (PGE2) is associated with phorbol ester-induced skin irritation and tumour promotion, but the mechanism of action is not fully understood and the role of keratinocyte-derived PGE2 is unclear. PGE2 was recently reported to modulate keratinocyte differentiation and phorbol-12-myristate-13-acetate (PMA), the most extensively studied phorbol ester tumour promoter in mouse skin, was shown to stimulate PGE2 release in human keratinocytes. Preliminary data on PGE2 release induced by PMA, mezerein, anthralin, sodium dodecyl sulphate and acetic acid in human keratinocyte cultures is compared to their response in rat keratinocytes. Our data confirms a previously published report on stimulation of PGE2 release by PMA in human keratinocytes and also demonstrates a difference in the magnitude of the PMA- and mezerein-induced response between human and rat keratinocyte cultures at non-cytotoxic concentrations. Cytotoxicity was evaluated by the Neutral Red uptake assay and a concentration that reduced cell viability to 50% of control was selected as a maximum concentration for subsequent measurement of PGE2 release. In contrast, anthralin, sodium dodecyl sulphate and acetic acid induced a similar degree of PGE2 release in human and rat keratinocyte cultures, but release was specifically associated with a cytotoxic response. Non-cytotoxic concentrations of these three chemicals did not stimulate release of PGE2. This study illustrates that PGE2 dose-response curves may reflect different mechanisms of action that may be intimately associated with skin irritant and tumour promoting activity. The data indicates a possible species difference in keratinocyte response to PMA and mezerein. The important value of keratinocyte cultures for mechanistic studies of tumour promotion and skin irritation is highlighted and further research is warranted into the potential role of intracellular pathways, which modulate keratinocyte differentiation and proliferation, in these processes.

Carcinogen-induced nuclear enlargement has been reported both in vitro and in vivo, but the mechanism, and whether it is causally related to carcinogenesis, has not yet been established. This study was designed to investigate the role of increased DNA content, such as might occur in polyploidy, in induction of nuclear enlargement. The effects of two genotoxic carcinogens, N-methyl-N-nitrosourea and adriamycin, were compared with the effects induced by diethylstilboestrol, which is arguably a non-genotoxic carcinogen but is known to induce polyploidy. HeLa S3 cells were used as the model system for comparison with previous studies. N-methyl-N-nitrosourea and adriamycin both induced a concentration-related increase in nuclear size 24 to 72 hr after a 30 min pulse-treatment. This was accompanied by an increase in the proportions of cells in the G(2) + M stage of the cell cycle, possibly due to a G(2) block. There was some evidence of polyploidy with adriamycin but not with N-methyl-N-nitrosourea. The distributions of nuclear areas indicated that increases in ploidy contributed to, but did not totally account for, the nuclear enlargement. In contrast, diethylstilboestrol increased the range of nuclear areas and DNA content, to both less than and greater than that of control cells, but only after a prolonged exposure period of 48 hr. These data were consistent with diethylstilboestrol inducing spindle damage. These results demonstrate that carcinogen-induced nuclear enlargement is only partially explained by increased nuclear DNA content, and that certain classes of non-genotoxic carcinogen may produce a completely different pattern to that from genotoxic carcinogens.

This study investigated the possible mechanism by which dichlorvos may have caused forestomach tumours in mice in a chronic corn oil gavage cancer bioassay [NTP (1989) Toxicology and carcinogenesis studies of dichlorvos in F344/N rats and B6C3F1 mice (gavage studies). National Toxicology Program Technical Report 342, NIH Publ. No 89-2598]. For this purpose, a method has been developed to assess the genotoxicity of irritant substances on mouse forestomach epithelium. Groups of five B6C3F1 mice were given a single oral dose of dichlorvos, the genotoxic forestomach carcinogen 1-methyl-3-nitro-1-nitrosoguanidine (MNNG) or the irritant, non-genotoxic forestomach carcinogen butylated hydroxyanisole (BHA). After periods of 2-48 h, three parameters were assessed: unscheduled DNA synthesis (UDS) by autoradiography of tissue sections, replicative DNA synthesis (RDS) also by autoradiography of incorporated [3H]thymidine, and histopathological changes, including hyperplasia. MNNG induced UDS but not RDS or hyperplasia in forestomach epithelium, consistent with its genotoxic mode of action. BHA and dichlorvos did not induce UDS, consistent with absence of genotoxic activity in the forestomach after in vivo exposure. In contrast, BHA and dichlorvos induced RDS and subsequent hyperplasia, which is likely to result from irritant damage. These data suggest that the chronic effects of dichlorvos on mouse forestomach epithelium in the oral gavage bioassay were mediated via enforced cell proliferation, rather than by a genotoxic mechanism.

Airborne contaminants have been implicated as important contributing factors in the aetiology of tumours of the respiratory tract. A major concern is that the potential carcinogenic activity of the particulate matter may be enhanced by the volatile material, which is adsorbed onto the surface. The cytotoxic potential of complex particulates was evaluated using the Neutral Red uptake method in primary cultures of hamster tracheal epithelial cells. Cytotoxicity of the reference particulates (benzo[a]pyrene adsorbed on to the surface of alumina A, alumina B, ferric oxide and titanium dioxide) was increased when cultures were treated with adsorbed rather than desorbed (plus dimethyl sulfoxide) samples. Foundry fume samples did not show the same effect.

The effect of the antihistaminic drug methapyrilene (MP) on DNA synthesis in primary cultures of rat hepatocytes has been compared with the effects of sodium phenobarbitone (PB), clofibric acid (CA), 2-acetylaminofluorene (AAF) and dimethyl sulfoxide (DMSO). The response for all chemicals was dependent on the concentration of epidermal growth factor (EGF) in the culture medium. MP at concentrations between 0.1 and 1 mum stimulated DNA synthesis. PB had a stimulatory effect on DNA synthesis at 0.5-1 mm. A greater increase in DNA synthesis was observed in the absence of EGF in the culture medium, for both chemicals. CA (0.1 mm) increased DNA synthesis in the absence of EGF and inhibited DNA synthesis at concentrations of 10 ng/ml or more. A dose-related increase in DNA synthesis with DMSO was observed in the presence of 10 ng EGF/ml. AAF did not stimulate DNA synthesis and inhibited it in the presence of 10 ng EGF/ml.

12 chemicals were selected for cytotoxicity testing, based on a report of a collaborative study (EC Project 86E3-063/86-460) that compared in vitro cytotoxicity in animal cell models with animal irritation and non-invasive human in vivo data. Neutral red (NR) and acid phosphatase (AP) activity were chosen as endpoints of cytotoxicity. AP activity has been shown to be a specific indicator of cytotoxicity in keratinocytes. NR(50) values (concentration that produces a 50% reduction in NR uptake compared with controls) and AP((peak)) values (concentration at which a peak activity occurs) were calculated for each chemical. Both in vitro assays exhibited good correlations with the animal irritation data. Poor correlations were obtained for comparison of the cutaneous blood flow volume data. The data presented provide useful information on the relationship between in vivo skin irritation and in vitro experimental models.

Biochemical parameters of cytotoxicity, such as the release of intracellular enzymes, appear to be useful for classification of irritant substances following in vivo chemical insult to the skin. Changes in activity of acid phosphatase (AP), a lysosomal enzyme, appear to parallel the development of the inflammatory response in laboratory animals after treatment with the known skin irritant sodium dodecyl sulphate (SDS). Neutral red (NR) uptake and AP were chosen as endpoints of cytotoxicity. NR and AP were measured in cultures of human epidermal keratinocytes, prepared from separate individuals, and in 3T3 cells following treatment with SDS. The NR(50) value (concentration producing a 50% reduction in NR uptake compared with controls) was similar in both cell types. AP in human keratinocyte cultures exhibited a peak activity, before declining at higher concentrations. This phenomenon was time dependent and was observed within 4 hr of treatment, but was not evident after a 24-hr exposure. The peak produced in 3T3 cells was negligible in comparison. The rate of NR uptake was also studied within the first 4 hr of exposure to SDS, which was comparable to the earlier time points at which AP was determined. The degree of inhibition of NR uptake was greater in human keratinocyte cultures than in 3T3 cells and a response was also elicited at lower dose levels in keratinocytes. AP may be a sensitive indicator of cytotoxicity in human keratinocytes and the data may be interpreted in relation to changes in lysosomal membrane function. This assay may be of value in assessment of skin irritation potential of aqueous soluble surfactants and chemicals that possess the ability to damage biological membranes.

The most common pathological response of the skin, associated with chemical-induced primary irritation, is inflammation. Release of inflammatory mediators occurs in primary irritant dermatitis in both laboratory animals and humans. Inflammatory mediators (prostaglandin E(2), leukotrienes C(4), D(4), E(4) and 15-hydroxyeicosatetranoic acid) were determined by radioimmunoassay, and leukotriene B(4) and the cytokine, interleukin 1alpha, were measured by enzyme-linked immunosorbent assay, in cultures of normal adult human keratinocytes. Release of inflammatory mediators into the culture medium was assessed at various time points following treatment of the cultures with the anionic surfactant sodium dodecyl sulphate (SDS), a known human skin irritant. A cytotoxic response was confirmed by the neutral red uptake assay after a 24-hr exposure to SDS. A dose-related release of inflammatory mediators was observed. The magnitude of the response varied between different mediators and as a function of time. The results show that release of inflammatory mediators occurs in human epidermal keratinocyte cultures, following chemical insult. Large inter-experimental variations in release of the various mediators probably preclude their use in a routine assay to determine irritation potential. In the case of SDS, release of inflammatory mediators is not a sensitive indicator of cytotoxicity. However, such techniques may provide important mechanistic data on the role of inflammatory mediators in the irritative response to certain chemicals, or on the possible role and interaction of certain mediators in the irritative process.

Skin is a major target organ for many experimental carcinogens that exist in our environment and the majority of previous carcinogenicity studies have utilised animal derived models. In view of the fact, that many of these environmental chemicals exhibit species- and tissue-specific metabolism, a human skin tissue derived model would be a distinct advantage. Squamous epithelial carcinoma is a predominant form of skin cancer in man and, in theory, human epidermal keratinocytes present an appropriate target cell to employ as an in vitro system to study epidermal carcinogenesis. This report demonstrates the valuable potential of human keratinocyte cultures as a suitable model for mechanistic studies on factors which may influence DNA damage and, hence, the subsequent development of cancer in human epidermis. Keratinocytes were serially cultivated from adult human skin samples and maintained in culture for at least 3 passages. Tertiary cultures, isolated from 3 separate individuals, were exposed to the direct-acting experimental carcinogen, methyl methanesulphonate (CAS No. 66-27-3), and benzo[a]pyrene (CAS No. 50-32-8), which requires metabolic activation. DNA repair was assessed by a quantitative autoradiographic technique. Methyl methanesulphonate and benzo[a]pyrene both elicited a dose-related increase in unscheduled DNA synthesis in cultures prepared from each individual. Inter-individual variation in the response was observed for each chemical, but this was greater in the case of benzo[a]pyrene, which indicates inter-individual variation in both xenobiotic metabolism activity and DNA repair capacity.

The in vivo-in vitro rat hepatocyte DNA-repair assay is regarded as labour-intensive and time-consuming to perform. This has tended to impose limitations on its use as a routine procedure for assessing the potential genotoxicity of chemicals. We have developed a simple wedge-perfusion technique which enables hepatocytes to be isolated from several different rats simultaneously. Hepatocyte yield and metabolic capacity are comparable to those isolated by conventional whole-liver perfusion. Hepatocyte viability was generally superior to that obtained when performing multiple in situ perfusions for the rat hepatocyte UDS assay. The median lobe is routinely used but no difference was observed in the UDS response to the positive control genotoxic agents, methyl methanesulphonate (MMS, CAS No. 66-27-3) and 2-acetylaminofluorene (AAF, CAS No. 53-96-3), in hepatocytes isolated from the median or either lateral lobe. The use of Williams medium E or Leibovitz L15 culture medium did not influence the response. This perfusion technique greatly reduces the time, equipment and personnel required and therefore the cost for hepatocyte isolation. It also facilitates the inclusion of concurrent control groups at each time point of assay.

Rat tracheal epithelial cells were cultured for 72 hr and then exposed for 3 hr to a range of test chemicals, including non-carcinogens, and direct-acting and activation-dependent carcinogens. The cells were then washed and cultured in fresh medium without the test chemicals for a further 24, 72 or 120 hr. Nuclear size measurements were then made. At 72 and 120 hr after exposure, those cultures treated with the non-carcinogens pyrene and anthracene had distributions of nuclear area similar to those of the solvent controls. All cultures treated with activation dependent carcinogens (N-nitrosopyrrolidine, N-nitrosodimethylamine, benzo[a]pyrene, dimethylbenz[a]anthracene or 4-nitroquinoline-N-oxide) or direct-acting carcinogens (nitrogen mustard or methylmethanesulphonate) showed a shift in distributions to the right, indicating enlarged nuclei. These results indicate that carcinogen-induced nuclear enlargement can occur in cultures of rat primary tracheal epithelial cells, and that this may be a useful indicator system for respiratory carcinogens.

Human keratinocytes are the most appropriate target cells to employ in an in vitro model to study the potential ability of a chemical to react with the genome of human skin cells. The resulting damage to DNA may be a key initiating factor in the subsequent development of squamous epithelial carcinoma, which is a common form of cancer in humans. Keratinocytes were routinely established from adult human skin samples and maintained in culture for at least three passages. Genotoxic damage was assessed by a quantitative autoradiographic technique which enables detection of unscheduled DNA synthesis without prior treatment of the cultures with agents, such as hydroxyurea, to suppress S-phase synthesis. Tertiary cultures from two individuals were exposed to the experimental carcinogens, methyl methane sulphonate (direct-acting genotoxin) and benzo[a]pyrene (requiring metabolic activation). Both chemicals induced unscheduled DNA synthesis in human keratinocytes. This study indicates that human keratinocyte cultures are a suitable model for the detection of genotoxic potential in human epidermis.

Adult rat hepatocytes, of good viability, were routinely isolated using a versatile biopsy perfusion technique. In order to develop an optimal cryopreservation regimen we investigated a variety of factors including the cooling rate, the constituents of the medium, the cryoprotective agents and the thawing conditions. The preferred freezing medium consisted of Leibovitz L15 culture medium supplemented with 10% (v/v) foetal calf serum, 10% (v/v) tryptose phosphate broth, 20% (v/v) dimethylsulphoxide, 100 mug/ml streptomycin and 100 U/ml penicillin. A freezing rate faster than the generally used 1-2 degrees C/min appeared to be optimal for subsequent hepatocyte attachment and survival in culture. Handling conditions after thawing were important for maximal recovery of cells in culture. To assess the functional integrity of hepatocytes in monolayer culture after cryogenic storage a number of parameters were monitored. Biochemical assays included lactate dehydrogenase leakage, protein synthesis, cellular ATP and ADP content, glutathione (reduced form), cytochrome P-450 content, ethoxycoumarin-O-deethylase activity, glucuronide and sulphate conjugation capacity. Cytochrome P-450 content was maintained at levels comparable with those in freshly prepared cultures for up to 24 hr in culture; ethoxycoumarin-O-deethylase activity was also similar to that of freshly prepared cultures. Changes in various biochemical parameters indicated that cryopreservation resulted in subtle damage to hepatocytes and that even cells that excluded dye did not survive as well as non-frozen cells. When human hepatocytes were subjected to the method developed for rat hepatocytes, they survived the trauma of cryopreservation but fewer cells subsequently attached and spread out to form a monolayer culture. These results indicate that human hepatocytes are more sensitive to freezing under the conditions developed for rat hepatocytes. This may reflect a greater sensitivity of the cells to cryopreservation and/or that the cryogenic conditions were not optimal for human hepatocytes.

An in vitro method has been established for the isolation and culture of tracheal epithelial cells for the evaluation of chemically induced genotoxicity using an unscheduled DNA synthesis assay. Cell cultures were derived from the Wistar albino rat and the golden Syrian hamster. Epithelial cells were isolated with protease type XIV for 16 hr and allowed to attach for 24 hr on collagen-coated coverslips in multi-well plates. Cells were exposed to the experimental carcinogens benzo[a]pyrene (metabolism-dependent) and methylmethanesulphonate (direct acting) for 24 hr. Benzo[a]pyrene and methylmethanesulphonate induced DNA repair in cultures isolated from hamsters, whereas only methylmethanesulphonate induced unscheduled DNA synthesis in rat tracheal epithelial cells, thus indicating the lack of metabolic activation in the rat cultures. These results could indicate the suitability of this culture system for the evaluation of airborne carcinogens.

Enlarged nuclei have been observed frequently as an early carcinogen-induced change in both cultured cells and in target tissues in vivo. The purpose of this work was to examine the occurrence of nuclear enlargement in the upper respiratory tract of rats to provide further evidence of whether nuclear enlargement is a reliable marker of carcinogenesis, and if it could be used as a short-term test for respiratory carcinogens. Carcinogen-induced nuclear enlargement is best demonstrated in vivo when the tissue involved has been undergoing rapid replication. Male Wistar albino rats were simultaneously exposed to an atmosphere containing sulphur dioxide (which caused a hyperplastic response in the nasal cavity) and received an i.p. injection of a nitrosamine. Sections were prepared from the nasal cavity, and the nuclear areas of respiratory epithelial cells were measured. There were some increases in nuclear size 24 and 72 h after the start of treatment. The reversal of this effect 120 h after the start of treatment may have been due to the loss of the normal ciliated mucosal epithelium, and subsequent loss of metabolic capability.

One major criticism of cytotoxicity tests is the negligible capacity for metabolism of foreign compounds, which exists in most cell lines. We have investigated the use of an exogenous metabolizing system, comprising 9000 g supernatant of liver from Aroclor 1254-pretreated rats (S-9) and an NADPH regenerating system, in the kenacid blue test. BCL-D1 cells (a human embryonic lung finite cell line) appeared to be sensitive to the toxicity of active metabolites of several model toxins. Other cell lines appeared to be less sensitive but the effects varied with the cell line and the test compound. These differences may, in part, be due to differences in the intrinsic ability of different cell lines to detoxify the active metabolites. It is therefore possible to use this type of test system for mechanistic studies but results with unknown compounds should be treated with caution.

Surfactants are widely used and often cause irritation to human skin. Three groups of surfactants, the trimethylammonium bromides (cationic), sodium dodecyl sulphate (anionic) and the polyoxyethylene sorbitans (Tweens, nonionic) were tested on a rat keratinocyte line (RTE) and an established fibroblast line (3T3-L1) to assess their potential as models for skin irritancy testing. Acid phosphatase (AP) release seems to parallel the development of signs of irritation in vivo and therefore AP activity was assayed after 4 hours' treatment to give an early indication of toxicity. AP activity in RTE keratinocytes rose to a peak and fell sharply, whereas in 3T3-L1 it did not change with treatment. Therefore AP may be a specific indicator of toxicity in keratinocytes. Neutral red (NR) uptake and kenacid blue (KB) staining were both assayed after 3 days' treatment as an indicator of cell proliferation. RTE and 3T3-L1 were equally sensitive in terms of NR and KB-ID(50) values for the anionic and nonionic compounds; however, 3T3 was more sensitive to the cationic compounds.

Hepatocyte structural and functional integrity was characterized in short-term primary monolayer cultures. Ethoxycoumarin-O-deethylase activity, cellular glutathione, ATP and ADP content, protein synthesis and lactate dehydrogenase (LDH) leakage were monitored. Cytotoxicity and perturbation of hepatocyte function involving measurement of these biochemical parameters was investigated following exposure to paracetamol and cyclophosphamide. Evaluation of several biochemical parameters appears to be a more appropriate assay of a perturbation in cellular integrity than the use of a single parameter. In the case of paracetamol the response of human hepatocyte cultures was broadly similar to that observed in rat hepatocyte cultures. An increase in LDH leakage occurred following exposure to high dose levels and was associated with depletion of cellular glutathione levels. At both toxic and non-toxic dose levels protein synthesis was impaired and a decrease in the cellular ATP/ADP ratio was evident. A decrease in protein synthesis and cellular glutathione was observed in both rat and human hepatocytes following exposure to cyclophosphamide. The data highlight the potential use of hepatocyte cultures for investigation of specific cytotoxic events and also emphasize the incorporation of human tissue in such systems.

1. The addition of 9000 g supernatant of rat liver homogenate (S9) or rat liver microsomal fractions to a cytotoxicity test system using BCL-D1 cells has been investigated. 2. The choice of culture medium influenced the intrinsic cytotoxicity of the metabolising system to the BCL-D1 cells. Use of Ham's F10 nutrient mixture resulted in greater cytotoxicity compared with several other media. 3. Microsomal fractions provided greater cytochrome P-450 dependent activation of cyclophosphamide and were less cytotoxic than S9. 4. Direct-acting toxic compounds, such as p-aminophenol, were less toxic in the presence of a metabolising system. This was due to protein-binding rather than enzymic detoxification.

Different methods of measuring cytotoxicity have been investigated in order to establish a test system for assessment of the toxicity of water samples. Ideally this should be highly sensitive and rapid to perform. Four variations of the neutral red uptake test have been compared with the MTT test and ATP determination. Chinese hamster V79/4 cells were used as the test system with 10 model toxins. All tests gave essentially similar results, with linear regression analysis producing correlation coefficients in excess of .93. No single test was most sensitive to all 10 compounds. The neutral red uptake and MTT tests could be performed in a single working day (approximately 8 h) if test compound and cells were plated out simultaneously. These tests are preferable to ATP determination, which is a complex and lengthy procedure, requiring expensive reagents.

Betamethasone and alpha-naphthoflavone produced similar activation of biphenyl 2-hydroxylase and benzo[a]pyrene 3-hydroxylase in control male rat liver microsomes. In small intestinal epithelial microsomes, betamethasone had no effect whereas alpha-naphthoflavone caused a pronounced activation of benzo[a]pyrene hydroxylation and a lesser activation of biphenyl 2-hydroxylation. In lung microsomes, betamethasone had no effect on either enzyme activity whereas alpha-naphthoflavone had no effect on biphenyl 2-hydroxylase but inhibited benzo[a]pyrene hydroxylase. In kidney cortex microsomes from male rats both compounds caused inhibition or had no effect whereas in kidney cortex microsomes female rats betamethasone activated whereas alpha-naphthoflavone had no effect. Activation also occurred in isolated viable hepatocytes from male rats. The response of biphenyl 2-hydroxylase was very similar to that found in male rat liver microsomes but benzo[a]pyrene hydroxylase was more sensitive to activation and less sensitive to inhibition than in microsomes. The findings are interpreted as demonstrating the presence of more than one 'latent' aromatic hydrocarbon hydroxylase in rodents.

1. 4-Hydroxylation was a major route of biphenyl metabolism in liver microsomes from control and phenobarbitone-pretreated rats, with 2- and 3-hydroxybiphenyl as lesser metabolites. 2. Many corticosteroids, when added to the microsomal incubation mixture, selectively increased 2-hydroxylation with little or no effect on 3- and 4-hydroxylation. Betamethasone caused the greatest activation (400%). 3. In liver microsomes from controls hamsters and 3-methylcholanthrene-pretreated rats, the basal hydroxylase activity, especially 2-hydroxylation, was much higher, but the quantitative increase following betamethasone addition was similar to that in liver microsomes from control and phenobarbitone-pretreated rats. 4. Pretreatment of rats with betamethasone also resulted in a small increase in biphenyl 2-hydroxylation activity after 4 h, returning to control values after 6 h. 5. In vitro addition of estradiol or testosterone had no effect on either basal or betamethasone-activated biphenyl 2-hydroxylation.

A differentiating keratinocyte cell line derived from explant cultures of rat sublingual epithelium has been used as a potential in vitro model for topical (skin) irritation of a range of detergents. The end points used to assess toxicity were acid phosphatase (AP) activity after 4 h of dosing and neutral red (NR) uptake and kenacid blue (KB) staining after 3 d to assess cell viability and number. The acid phosphatase activity increased to a sharp peak with increasing doses, then fell equally sharply for the anionic detergent sodium dodecyl sulfate (SDS) and cationic detergents TMABs (trimethylammonium bromides). With the Tweens (the least toxic group) there was no acid phosphatase peak or it appeared at the highest dose level used (1.0 mg/ml). The dose-response curves for NR uptake paralleled those for KB staining. With all of the three end points, it was apparent that the order of toxicity for the different groups was TMABs greater than SDS greater than Tweens, with a difference of one order of magnitude between consecutive groups when using NR and KB.

In order to establish the mechanism by which the selective activation of biphenyl 2-hydroxylation by betamethasone occurs the effect of modifying possible critical factors in the hydroxylation process has been examined. Activation of biphenyl 2-hydroxylation by betamethasone was found in detergent-solubilized rat liver microsomes indicating that intact microsomal membranes are probably not necessary for the activation. Betamethasone had no effect on the spectrally apparent binding of biphenyl or of other type I, type II or reverse type I model substrates. The activation process did not appear to be greatly influenced by changing the ratio of cytochrome P-450 reductase to cytochrome P-450 nor by changing the amount of NADPH. Addition of NADH increased the extent of activation suggesting that betamethasone facilitates transference of the second electron to cytochrome P-450. However, betamethasone also stimulated cumene hydroperoxide supported biphenyl 2-hydroxylation; therefore a step subsequent to cytochrome P-450 reduction is also involved in the activation. Activation did not correlate with increased uncoupling of an active oxygen-cytochrome P-450 complex to form hydrogen peroxide.