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There is quite an extensive set of epidemiology studies conducted for a range of different radiation exposure scenarios and in some cases at doses that can be considered to be in the low dose range (

This review is a contribution to the memory of Dr William (Bill) Morgan and highlights an area of research and deliberation that he considered extremely important in support of the setting of protective radiation dose limits. Biological research has generally played a minor role in the estimation of adverse health outcomes following exposure to low doses and low dose rates of radiation. The reliance has been on the available, quite extensive data base of epidemiology studies. The major concern is that such studies are for moderate to high doses requiring risk extrapolation methodologies for estimating low dose effects. There are significant uncertainties associated with this approach. This review will discuss how radiation biology studies can potentially reduce this uncertainty through the use of a key events/adverse outcome pathways approach to identify bioindicators of cancer and non-cancer effects for use as parameters in biologically-based dose-response (BBDR) models. Such models would allow for an improved extrapolation approach for estimating health effects at low doses and low dose rates of radiation.Based on reported and ongoing studies for environmental chemicals, the adverse outcome/key events approach is a viable one for enhanced risk assessment (and risk management practice). The identification of informative bioindicators of adverse health effects will be a challenge but with modern molecular and advanced computational techniques, it is certainly feasible. This approach provides a framework for defining a low dose radiation research program; something that was of great importance to Bill Morgan.

A public workshop, organized by a Steering Committee of scientists from government, industry, universities and research organizations, was held at the National Institute of Environmental Health Sciences (NIEHS) in September, 2010. The workshop explored the dose-response implications of toxicant modes of action (MOA) mediated by nuclear receptors. The dominant paradigm in human health risk assessment has been linear extrapolation without a threshold for cancer, and estimation of sub-threshold doses for non-cancer and (in appropriate cases) cancer endpoints. However, recent publications question the application of dose-response modeling approaches with a threshold. The growing body of molecular toxicology information and computational toxicology tools has allowed for exploration of the presence or absence of sub-threshold doses for a number of receptor-mediated MOAs. The workshop explored the development of dose-response approaches for nuclear receptor-mediated liver cancer, within a MOA Human Relevance Framework (HRF). Case studies addressed activation of the AHR, the CAR and the PPARα. This article describes the workshop process, key issues discussed and conclusions. The value of an interactive workshop approach to apply current MOA/HRF frameworks was demonstrated. The results may help direct research on the MOA and dose-response of receptor-based toxicity, since there are commonalities for many receptors in the basic pathways involved for late steps in the MOA, and similar data gaps in early steps. Three additional papers in this series describe the results and conclusions for each case-study receptor regarding its MOA, relevance of the MOA to humans and the resulting dose-response implications.

Purpose: This review evaluates the role of dose rate on cell and molecular responses. It focuses on the influence of dose rate on key events in critical pathways in the development of cancer. This approach is similar to that used by the U.S. EPA and others to evaluate risk from chemicals. It provides a mechanistic method to account for the influence of the dose rate from low-LET radiation, especially in the low-dose region on cancer risk assessment. Molecular, cellular, and tissues changes are observed in many key events and change as a function of dose rate. The magnitude and direction of change can be used to help establish an appropriate dose rate effectiveness factor (DREF). Conclusions: Extensive data on key events suggest that exposure to low dose-rates are less effective in producing changes than high dose rates. Most of these data at the molecular and cellular level support a large (2–30) DREF. In addition, some evidence suggests that doses delivered at a low dose rate decrease damage to levels below that observed in the controls. However, there are some data human and mechanistic data that support a dose-rate effectiveness factor of 1. In summary, a review of the available molecular, cellular and tissue data indicates that not only is dose rate an important variable in understanding radiation risk but it also supports the selection of a DREF greater than one as currently recommended by ICRP (2007) and BEIR VII (NRC/NAS 2006).

The U.S. Environmental Protection Agency's Guidelines for Carcinogen Risk Assessment encourages the use of mechanistic data in the assessment of human cancer risk at low (environmental) exposure levels. The key events that define a particular mode of action for tumor formation have been concentrated to date more on mutational responses that are broadly the result of induced DNA damage and enhanced cell proliferation. While it is clear that these processes are important in terms of tumor induction, other modes that fall under the umbrella of epigenetic responses are increasingly being considered to play an important role in susceptibility to tumor induction by environmental chemicals and as significant modifiers of tumor responses. Alterations in gene expression, DNA repair, cell cycle control, genome stability and genome reprogramming could be the result of modification of DNA methylation and chromatin remodeling patterns as a consequence of exposure to environmental chemicals. These concepts are described and discussed.

The US Environmental Protection Agency recently released its new guidelines for carcinogen risk assessment together with supplemental guidance for assessing susceptibility from early-life exposure to carcinogens. In particular, these guidelines encourage the use of mechanistic data in support of dose-response characterization at doses below those at which an increase in tumor frequency over background levels might be detected. In this context of the utility of mechanistic data for human cancer risk assessment, the International Life Sciences Institute (ILSI) has developed a human relevance framework (HRF) that can be used to assess the plausibility of a mode of action (MoA) described for animal models operating in humans. The MoA is described as a sequence of key events and processes that result in an adverse outcome. A key event is a measurable precursor step that is in itself a necessary element of the MoA or is a bioindicator for such an element. A number of cellular and molecular perturbations have been identified as key events whereby DNA-reactive chemicals can produce tumors. These include DNA adducts in target tissues, gene mutations and/or chromosomal alterations in target tissues and enhanced cell proliferation in target tissues. This type of data integration approach to quantitative cancer risk assessment can be applied to 1,3-butadiene, for example, using data on biomarkers in exposed Czech workers [1]. For this study, an extensive range of biomarkers of exposure and response was assessed, including: polymorphisms in metabolizing enzymes; urinary concentrations of several metabolites of 1,3-butadiene; hemoglobin adducts; HPRT mutations in T-lymphocytes; chromosomal aberrations by FISH and conventional staining procedures; sister chromatid exchanges. Exposure levels were monitored in a comprehensive fashion. For risk assessment purposes, these data need to be considered in the context of how they inform the MoA for leukemia, the tumor type reported to be increased in synthetic rubber workers exposed to 1,3-butadiene. Also, for the HRF it is necessary to establish key events for a MoA in rodents for the induction of tumors by 1,3-butadiene. There is clearly a species difference in sensitivity to tumor induction, with mice being much more sensitive than rats; key events need to explain this difference. For butadiene, the MoA is DNA-reactivity and subsequent mutagenicity and so following the EPA's cancer guidelines, a linear extrapolation is used from the point of departure (POD), unless additional data support a non-linear extrapolation. For the present case, the human bioindicator data are not informative as far as dose-response characterization is concerned. Mouse chromosome aberration data for in vivo exposures might be used for establishing a POD, with linear extrapolation from this POD. The available cytogenetic data from rodent studies appear to be sufficiently extensive and consistent for this to be a viable approach. This approach of using MoA and key events to establish the human relevance can lead to the development of specific informative bioindicators of response that can be used as surrogates to predict the shape of the tumor dose response curve at low doses. Truly informative predictors of tumor responses should be able to provide estimates of human tumor frequencies at low, environmental exposures to 1,3-butadiene.

Recognition that children are a potentially susceptible subpopulation has led to the development of child-specific sensitivity factors. Establishing reliable sensitivity factors in support of risk assessment of early-life stage exposures can be aided by evaluating studies that enhance our understanding both of the biological basis of disease processes and the potential role of environmental exposures in disease etiology. For these reasons, we evaluated childhood acute lymphocytic leukemia (ALL) studies from the point of view of mechanism and etiology. ALL is the most common form of childhood cancer proposed to result from a prenatal primary event and a postnatal second event. This multi-stage model is supported by the observation that chromosomal translocations/fusion genes (e.g., TEL-AML1) involved in producing ALL are detected at birth (prenatal event), and a postnatal event (e.g., TEL deletion) is required for disease manifestation. It appears that a proportion of ALL cases are the result of environmental exposures, in which case preconceptional, prenatal, and postnatal stages are likely to be critical exposure windows. To this end, we recognized postnatal infection-related risk factors as potential candidates associated with the ALL second event. Additionally, we discuss use of ALL-associated fusion genes and genetic polymorphisms, together or separately, as indicators of ALL susceptibility and increased risk. The possibility of using fusion genes alone as biomarkers of response is also discussed because they can serve as predictors of key events in the development of a mode of action (a sequence of key events, starting with interaction of an agent with a cell, ultimately resulting in cancer formation) for particular environmental exposures. Furthermore, we discuss use of an initiated animal model for ALL, namely transgenic mice with TEL-AML1 expression, for exploring mechanisms by which different classes of environmental exposures could be involved in inducing the postnatal step in ALL formation.

It has been known for decades that mutagenicity plays an important role in the activity of most carcinogens. This mutagenicity can result from direct damage to DNA through a chemical being DNA reactive or from indirect effects, such as through the production of oxygen radicals that then react with DNA. This article presents a set of key events whereby DNA reactivity initiates the process of carcinogenicity that leads to the subsequent mutation induction and enhanced cell proliferation that ultimately results in tumor development. This set of key events for DNA-reactive chemicals was applied to two case studies (aflatoxin B1 and dichloromethane) with the aim of assessing the utility of the Human Relevance Framework (HRF) for this class of chemicals. The conclusions were that the HRF was a viable approach for the use of mechanistic data for DNA-reactive chemicals obtained from both laboratory animals and human cells in vivo and in vitro for predicting human carcinogenicity. In the case of aflatoxin B1, the HRF could be used to predict that carcinogenicity in humans was a likely outcome. In contrast, the HRF predicted that the human carcinogenic potential of dichloromethane was at best less likely than in rodents; this conclusion was supported by the available epidemiological data.

A complete mode of action human relevance analysis--as distinct from mode of action (MOA) analysis alone--depends on robust information on the animal MOA, as well as systematic comparison of the animal data with corresponding information from humans. In November 2003, the International Life Sciences Institute's Risk Science Institute (ILSI RSI) published a 2-year study using animal and human MOA information to generate a four-part Human Relevance Framework (HRF) for systematic and transparent analysis of MOA data and information. Based mainly on non-DNA-reactive carcinogens, the HRF features a "concordance" analysis of MOA information from both animal and human sources, with a focus on determining the appropriate role for each MOA data set in human risk assessment. With MOA information increasingly available for risk assessment purposes, this article illustrates the further applicability of the HRF for reproductive, developmental, neurologic, and renal endpoints, as well as cancer. Based on qualitative and quantitative MOA considerations, the MOA/human relevance analysis also contributes to identifying data needs and issues essential for the dose-response and exposure assessment steps in the overall risk assessment.

The HESI RISK21 project formed the Dose-Response/Mode-of-Action Subteam to develop strategies for using all available data (in vitro, in vivo, and in silico) to advance the next-generation of chemical risk assessments. A goal of the Subteam is to enhance the existing Mode of Action/Human Relevance Framework and Key Events/Dose Response Framework (KEDRF) to make the best use of quantitative dose-response and timing information for Key Events (KEs). The resulting Quantitative Key Events/Dose-Response Framework (Q-KEDRF) provides a structured quantitative approach for systematic examination of the dose-response and timing of KEs resulting from a dose of a bioactive agent that causes a potential adverse outcome. Two concepts are described as aids to increasing the understanding of mode of action-Associative Events and Modulating Factors. These concepts are illustrated in two case studies; 1) cholinesterase inhibition by the pesticide chlorpyrifos, which illustrates the necessity of considering quantitative dose-response information when assessing the effect of a Modulating Factor, that is, enzyme polymorphisms in humans, and 2) estrogen-induced uterotrophic responses in rodents, which demonstrate how quantitative dose-response modeling for KE, the understanding of temporal relationships between KEs and a counterfactual examination of hypothesized KEs can determine whether they are Associative Events or true KEs.

Non-genotoxic chemical carcinogens are capable of inducing tumors in rodents without interacting with or directly altering the genetic material. Since a preponderance of evidence suggests that cancer results from the accumulation of genetic alterations, the mechanisms by which many non-genotoxic carcinogens induce genotoxic events remain unclear. The present study investigated whether the mitogenic, non-genotoxic carcinogen phenobarbital (PB) could alter cell-cycle checkpoint controls, thereby indirectly leading to the accumulation of genetic damage. Initial studies involved characterizing cell-cycle checkpoint responses to DNA damage in freshly isolated B6C3F1 mouse hepatocytes. These cells responded to bleomycin-induced DNA damage by arresting in G1 and G 2 . Cell-cycle arrest was coupled with p53 protein induction ; however, p21 WAFI protein levels remained unchanged. Studies that utilized hepatocytes isolated from C57BL p53 -/- mice showed that the DNA damage-induced G 1 cell-cycle arrest was dependent on p53 function, but cell-cycle arrest in G 2 was not affected by loss of p53. PB was able to delay and attenuate the G 1 checkpoint response without altering G 2 checkpoint function. A reduction in p53 protein, but not transcript levels, was observed in hepatocytes exposed to PB. Additionally, PB delayed and attenuated p53 protein induction during DNA damage, which suggests that changes in the p53 protein may be contributing to the attenuated G 1 checkpoint response caused by PB. Altered G 1 checkpoint function represents an epigenetic mechanism by which phenobarbital may prevent the detection and repair of DNA damage and indirectly increase the frequency of genotoxic events above that occurring spontaneously. Abrogation of checkpoint controls may, thus, play an important mechanistic role in mitogenic, non-genotoxic chemical carcinogenesis.

The information for the present discussion on the uncertainties associated with estimation of radiation risks and probability of disease causation was assembled for the recently published NCRP Report No. 171 on this topic. This memorandum provides a timely overview of the topic, given that quantitative uncertainty analysis is the state of the art in health risk assessment and given its potential importance to developments in radiation protection. Over the past decade the increasing volume of epidemiology data and the supporting radiobiology findings have aided in the reduction of uncertainty in the risk estimates derived. However, it is equally apparent that there remain significant uncertainties related to dose assessment, low dose and low dose-rate extrapolation approaches (e.g. the selection of an appropriate dose and dose-rate effectiveness factor), the biological effectiveness where considerations of the health effects of high-LET and lower-energy low-LET radiations are required and the transfer of risks from a population for which health effects data are available to one for which such data are not available. The impact of radiation on human health has focused in recent years on cancer, although there has been a decided increase in the data for noncancer effects together with more reliable estimates of the risk following radiation exposure, even at relatively low doses (notably for cataracts and cardiovascular disease). New approaches for the estimation of hereditary risk have been developed with the use of human data whenever feasible, although the current estimates of heritable radiation effects still are based on mouse data because of an absence of effects in human studies. Uncertainties associated with estimation of these different types of health effects are discussed in a qualitative and semi-quantitative manner as appropriate. The way forward would seem to require additional epidemiological studies, especially studies of low dose and low dose-rate occupational and perhaps environmental exposures and for exposures to x rays and high-LET radiations used in medicine. The development of models for more reliably combining the epidemiology data with experimental laboratory animal and cellular data can enhance the overall risk assessment approach by providing biologically refined data to strengthen the estimation of effects at low doses as opposed to the sole use of mathematical models of epidemiological data that are primarily driven by medium/high doses. NASA's approach to radiation protection for astronauts, although a unique occupational group, indicates the possible applicability of estimates of risk and their uncertainty in a broader context for developing recommendations on: (1) dose limits for occupational exposure and exposure of members of the public; (2) criteria to limit exposures of workers and members of the public to radon and its short-lived decay products; and (3) the dosimetric quantity (effective dose) used in radiation protection.

In the past few years, our knowledge of mammalian genomes has increased enormously. Our understanding of the molecular basis of the normal cellular processes of DNA replication and repair and cell cycle control, together with how their fidelity malfunctions as part of tumor development, has increased in parallel. This has led to a clearer appreciation that there are subpopulations that have been generically described as being genetically or otherwise susceptible to the induction of cancer or birth defects. The term susceptibility is a default option, since there clearly will be a very broad range of sensitivities among the so-called susceptible populations, dependent upon the specific underlying mechanism. This could lead to the conduct of risk assessments for each specific situation, involving both genotypes of individuals and agents of concern. This would ideally take into account the effects on response of various modifying factors, genetic and other. One advantage to be gained from this approach is the ability to determine if a particular susceptibility places subpopulations at extreme risk as compared to the overall normal distribution of risk in the population, or whether such a susceptible population presents a slight extension of the upper bound of the risk distribution or lies within the normal distribution. In addition, the specific mechanism of the susceptibility as related to exposure scenarios and the magnitude and demographics of the susceptible populations need to be taken into account. Thus, the management of risk has to be linked to the specific risk assessment. For many of the so-called susceptible populations an uncertainty factor of less than 10, even including 1, would be predicted to bring the risk within the normal distribution. It is hoped that as more mechanistic information on susceptibility becomes available and a specific risk can be defined, the practice of risk management will be considerably improved.

The involvement of p53 in the formation of chromosome aberrations was assessed by analyzing bleomycin-induced, chromatid-type aberrations in G 2 phase fibroblasts derived from embryos from wild-type and p53 knock-out mice. Cells that were p53+/- or p53-/- were more sensitive to the induction of aberrations than the p53+/+ cells, particularly at concentrations of 7.5 and 10.0 μg/ml. The p53-deficient cells also showed an overdispersed distribution of bleomycin-induced chromatid aberrations, a greater amount of overall genomic instability and a possible loss of a cell death pathway. These data are interpreted as indicating a role for p53 in DNA repair in the G 2 phase, with a loss of p53 leading to an increased frequency of deletions (incomplete repair) and interchanges (misrepair). The specific role remains to be elucidated. The mitotic index decreased with increasing bleomycin concentration to a similar extent in all three cell lines, indicating that the loss of a G 2 checkpoint in p53-/- and p53+/- cells was not an explanation for the increased sensitivities in these cells compared with the p53+/+.

Steers (n = 156) finished on rhizoma peanut (Arachis glabrata Benth.)-tropical grass pasture in Florida and slaughtered at Central Packing, Center Hill were compared with steers (n = 152) finished on a concentrate diet in Texas and slaughtered at Excel, Plainview. Average daily gain during the growing and finishing periods was lower (P.001) for forage-finished steers (.49 and .94 kg/d, respectively) than for concentrate-finished steers (.78 and 1.33 kg/d, respectively). Forage-finished steers had less fat over the ribeye (8.3 vs 11.4 mm; P.01), lighter hot carcass weight (280 vs 346 kg; P.001), and smaller longissimus muscle area (70.8 vs 86.6 cm2; P.001) than concentrate-finished steers. Yield grade was not different (2.7 vs 2.6; P.10), but quality grade was slightly better (low Select vs mid Select; P.01) for concentrate-finished steers. Lean color of forage-finished steers was darker (P.001) and fat of forage-finished steers had a creamier color (P.001), but carcasses were not discounted due to yellow fat color. Shear force values were higher (6.8 vs 4.0 kg; P.001) for forage-finished than for concentrate-finished steers. Off-flavors were detected by trained sensory panelists in 36% of forage-finished and 14% of concentrate-finished carcasses, but all at barely detectable levels. This research indicates that steers can be finished on rhizoma peanut-tropical grass pastures, but with some reduction in quality grade relative to concentrate-finished steers.(ABSTRACT TRUNCATED AT 250 WORDS)

Clinical investigations are studies designed to evaluate the effectiveness of a new animal drug. Expectations for documentation of events occurring during clinical investigations have been greatly increased. The Food and Drug Administration (FDA) through its Center for Veterinary Medicine (CVM) division recently issued a guideline to address the responsibilities (under 21 CFR 511.1 and 512[j] of the Federal, Food, Drug and Cosmetic Act) of investigators who conduct clinical investigations of new animal drugs and of monitors of these investigations. The guideline is part of a continuing effort by FDA/CVM to propose data integrity initiatives that will continue to assure the reliability and accuracy of the data upon which decisions to approve new animal drugs are based. In addition to the increased documentation, FDA/CVM intends to make real-time inspection of clinical investigations a routine practice. In response to these changes; those involved with clinical investigations will need to make appropriate adjustments. The purpose of this review is to provide additional notification to clinical investigators of the changes in their responsibilities under the new guideline and to provide an investigator perspective of how these changes might impact research efforts.

The US Environmental Protection Agency (EPA) developed a genetic risk assessment model for exposures to ethylene oxide utilizing data on the induction of reciprocal translocations in male germ cells [Rhomberg et al. 1990]. This particular approach served as a reasonable initial attempt, albeit somewhat limited with regard to endpoint and only partially attentive to the mechanisms of induction of genetic alterations and the behavior of chromosomes during meiosis. The present paper discusses the scientific basis for a reassessment of the EPA model, providing data and hypotheses related to effective dose to the target cells and shape of the dose-response relationship at low doses, and dose rates. While the present genetic risk assessment approach is discussed in terms of ethylene oxide, it would be applicable to most mutagenic chemicals. The outcome of the discussion is that the genetic risk for exposed males from reciprocal translocation induction will be negligible at low doses since the dose-response curve is likely to be a function of the square of the dose. In addition, the proportion of genetically unbalanced live born offspring in humans arising from reciprocal translocation carriers is less than 10% of the frequency formed through meiotic segregation and fertilization for such carriers. Simply from a consideration of mechanism—namely, the very high probability of DNA repair prior to the next S-phase for a resting oocyte—it would be predicted that there would be a very low to negligible frequency of translocations in female germ cells from ethylene oxide exposure. It is further stressed that additional components of a genetic risk model require a consideration of all germ cell stages in the male, and the inclusion of calculations for point and deletion mutations. Some indications of likely response are presented with these points in mind. © 1995 Wiley-Liss, Inc.

The effects of roughage level (10, 20, or 30% roughage equivalent [RE]), roughage source (alfalfa vs cottonseed hulls), roughage regimen (constant RE vs 2% RE during the mid-finishing period), tallow level (1.2 vs 4.6%), and steer type (British crossbred [BRITX] vs Bos indicus crosses [BRX]) were evaluated in three experiments with a common allotment and several overlapping treatments. Steers (n = 432; initial weight = 326 +/- 26 kg) were divided into three BW blocks and allotted randomly to 72 pens and 24 treatments. Steers were fed steam-flaked, sorghum grain-based finishing diets for 124 to 166 d. Diets with 20% RE decreased gain efficiency and 30% RE diets decreased both gain (linear, P.07) and efficiency (linear, P.001) compared with 10% RE diets. Reducing roughage level during the mid-finishing period improved overall gain efficiency 2, 7, and 24% (P.2,.05, and.001, respectively) for the 10, 20, and 30% RE diets, respectively. Steers fed cottonseed hulls consumed more feed (9.6 vs 8.8 kg/d, P.001) but tended to gain less (1.53 vs 1.58 kg/d, P = .11) than steers fed alfalfa, were leaner, and had fewer carcasses grading Choice (62 vs 77%, P.05). Feeding 4.6% tallow decreased DMI (P.05) and improved gain efficiency (P.05) compared with 1.2% tallow. The BRITX steers consumed more feed (6%, P.001) but were somewhat less efficient (3.5%, P.05) than BRX steers. Carcasses from BRITX steers tended to be fatter than carcasses from BRX steers and more of them graded Choice (62 vs 37%, P.01). Commercial BRX steers did not perform as well as BRITX steers on higher-energy-density diets (4.6% tallow or variable roughage regimen). Knowledge of the genetic background of feeder cattle can be important in the selection of dietary energy density and marketing expectations.

Four mutant strains of Paramecium tetraurelia with a reduced ability to respond behaviorally to Mg2+ have been isolated. Voltage-clamp analyses showed that their Mg2+ insensitivity is associated with a reduced Ca(2+)-dependent Mg2+ current. The four mutants, which have been doubled "eccentric," result from recessive mutations in two unlinked loci, xntA and xntB. Further analysis of xntA1 showed it to be unlinked to any of the behavioral mutants of P. tetraurelia described previously, but it is allelic to d4-521, a "K(+)-resistant" strain, and d4-596, a "Ba(2+)-shy" mutant. The varied pleiotropic effects of xntA1, which include increased resistance to Ni2+ and Zn2+ poisoning, suggest that the locus encodes a central regulator of cell function in Paramecium.

We investigated the effects of supplementing Rambouillet x Suffolk wether lambs grazing irrigated 'Cimarron' alfalfa (Medicago sativa L.) with three levels (0 [C0], 123 [C123], and 247 [C247] g of DM.lamb-1.d-1) of cracked corn. Each treatment group also received 190 g of a supplement designed to prevent bloat. Replicated pastures (three per treatment) grown on a fine, mixed, thermic Torretic Paleustoll soil were grazed rotationally (forage plus supplement allowance of 6.5% of BW/d) by lambs for 85 d during spring 1992. Supplemental corn levels were analyzed as single degree of freedom contrasts for linear and quadratic effects. At the start of the experiment, lambs weighed 30.7 +/- .32 kg. Average daily gains for C0 C123, and C247 were 141, 154, and 169 g/d, respectively. Lamb production per hectare increased quadratically (P.01) with increasing corn level (C0 [716 kg of lamb/ha], C123 [816 kg of lamb/ha], and C247 [964 kg of lamb/ha]). Supplementation with C247 vs C0 increased carcass weights (11%), dressing percentage (6%), and backfat thickness (30%). Plasma urea N (PUN) concentrations did not differ (P.10) between C0 and C123 after 27 d of corn supplementation, but after 75 d PUN concentrations between C0 and C123 had decreased (P.10) by 11%. For C247, PUN concentrations after 27 and 75 d of corn supplementation had decreased (P.10) by 17 and 18%, respectively, compared with C0. Plasma urea N concentrations increased (P.01) linearly (r2 = .93) with an increase in digestible CP:DE ratio (DP:DE).(ABSTRACT TRUNCATED AT 250 WORDS)

Clonal growth modeling of carcinogenesis requires data on the number of cells at risk of becoming cancerous. We synthesized literature data to estimate the fraction of respiratory tract epithelial cells that are progenitor cells, and therefore at risk, in formaldehyde carcinogenesis for specific respiratory tract regions. We concluded that the progenitor cells for the transitional and respiratory epithelia of the nose are basal and nonciliated cells and Type II cells in the alveolar region. In the conducting airways, our evaluation indicated that ciliated and basal cells are not in the progenitor pool. Respiratory tract epithelial cell fractions of 0.819 in rats and 0.668 in humans were estimated from the data. The total numbers of epithelial cells in the lower respiratory tract of humans and rats were allocated to individual generations. Cell cycle times were also estimated from literature data, since the reciprocal of cell cycle time is an important variable in clonal growth modeling. Sensitivity analyses of a previously published risk model for formaldehyde carcinogenesis showed that specification of the fraction of cells at risk markedly affects estimates of some parameters of the clonal growth model. When all epithelial cells are considered part of the progenitor pool, additional risks for the non-smoking population was typically over predicted by about 35% for high exposure levels. These results demonstrate the importance of accurately identifying cell populations at risk when applying quantitative models in risk assessments.

Groups of male B6C3F1 mice were exposed by inhalation to 0, 25, 50, 100 or 200 p.p.m. ethylene oxide (EO) for up to 48 weeks (6 hours/day, 5 days/week). Animals were sacrificed at 6, 12, 24 and 48 weeks after the start of the exposure for analyses of reciprocal translocations in peripheral blood lymphocytes and germ cells. The frequency of the total chromosomal aberrations in the peripheral blood lymphocytes was significantly increased at the 100 and 200 p.p.m. exposure concentrations at the 12-week time point, at 50, 100 and 200 p.p.m. at the 24-week time point and at all EO concentrations at the 48-week time point. The frequency of stable reciprocal translocations, which can be used as biomarkers, was increased (P < 0.05) at 100 and 200 p.p.m. at the 12-week time point, at 100 and 200 p.p.m. at the 24-week time point and at 50, 100 and 200 p.p.m. at the 48-week time point. No statistically significant increase could be observed in translocation frequencies at the 6-week time point in the peripheral blood lymphocytes. The exposure-response curves were non-linear when the frequencies of translocations were plotted against EO exposure durations or against EO exposure concentrations. There was no effect of exposure concentration rate on reciprocal translocation frequency. Reciprocal translocations induced in spermatogonial stem cells (observed at the sprematocyte stage) showed significant increases in translocation frequencies over controls at all EO concentrations at 48 weeks. However, increases were small and they did not occur in a dose-responsive manner. The statistically significant increase observed at 12 weeks in the spermatocytes was equivocal. This study provides low-level chronic exposure somatic cytogenetic data generated in mice that can be used to support the shape of the tumour dose-response in rodents and humans The germ cell cytogenetic data are discussed in terms of its relevance for a threshold response for genetic effects at low exposures.

The assessment of human cancer risk from chemical exposure requires the integration of diverse types of data. Such data involve effects at the cell and tissue levels. This report focuses on the specific utility of one type of data, namely DNA adducts. Emphasis is placed on the appreciation that such DNA adduct data cannot be used in isolation in the risk assessment process but must be used in an integrated fashion with other information. As emerging technologies provide even more sensitive quantitative measurements of DNA adducts, integration that establishes links between DNA adducts and accepted outcome measures becomes critical for risk assessment. The present report proposes an organizational approach for the assessment of DNA adduct data (e.g., type of adduct, frequency, persistence, type of repair process) in concert with other relevant data, such as dosimetry, toxicity, mutagenicity, genotoxicity, and tumor incidence, to inform characterization of the mode of action. DNA adducts are considered biomarkers of exposure, whereas gene mutations and chromosomal alterations are often biomarkers of early biological effects and also can be bioindicators of the carcinogenic process.

Epidemiological studies suggest that chronic exposure to inorganic arsenic is associated with cancer of the skin, urinary bladder and lung as well as the kidney and liver. Previous experimental studies have demonstrated increased incidence of liver, lung, ovary, and uterine tumors in mice exposed to 85 ppm (approximately 8 mg/kg) inorganic arsenic during gestation. To further characterize age susceptibility to arsenic carcinogenesis we administered 85 ppm inorganic arsenic in drinking water to C3H mice during gestation, prior to pubescence and post-pubescence to compare proliferative lesion and tumor outcomes over a one-year exposure period. Inorganic arsenic significantly increased the incidence of hyperplasia in urinary bladder (48%) and oviduct (36%) in female mice exposed prior to pubescence (beginning on postnatal day 21 and extending through one year) compared to control mice (19 and 5%, respectively). Arsenic also increased the incidence of hyperplasia in urinary bladder (28%) of female mice continuously exposed to arsenic (beginning on gestation day 8 and extending though one year) compared to gestation only exposed mice (0%). In contrast, inorganic arsenic significantly decreased the incidence of tumors in liver (0%) and adrenal glands (0%) of male mice continuously exposed from gestation through one year, as compared to levels in control (30 and 65%, respectively) and gestation only (33 and 55%, respectively) exposed mice. Together, these results suggest that continuous inorganic arsenic exposure at 85 ppm from gestation through one year increases the incidence and severity of urogenital proliferative lesions in female mice and decreases the incidence of liver and adrenal tumors in male mice. The paradoxical nature of these effects may be related to altered lipid metabolism, the effective dose in each target organ, and/or the shorter one-year observational period.

The objective of this study was to determine the minimum dosage of recombinant bovine somatotropin (bST) required to elicit maximum depression in plasma urea nitrogen (PUN), an indicator of anabolic activity. Twenty-four steers (389 kg) were blocked by weight into six pens. Six steers were placed on each of the following bST doses: 0, 8, 16 and 32 mg bST/d. Treatments were administered once daily via subcutaneous injections for 21 d. Steers were weighed and jugular blood samples were taken on d 0, 1, 4, 7, 10, 13, 16 and 21 at 1400, approximately 4 h after feeding. Delta PUN (DPUN) was calculated as PUN - d 0 PUN. There was no dose x time interaction (P = .94) in DPUN. Maximum reduction in DPUN with bST occurred by d 7 (P less than .05). Linear (P less than .01) and quadratic (P less than .05) orthogonal contrasts indicated that DPUN depression increased with bST administration, with maximal reduction calculated to occur with 23 mg (59 micrograms/kg) bST/d. There was no further decrease in DPUN with 32 than with 16 mg bST, indicating that the minimum daily dose is at least 16 mg but no more than 23 mg. A similar dose response was observed in daily gain. Results from this study indicate that bST reduced PUN in a dose-dependent manner and that 41 to 64 micrograms/kg body weight maximized the anabolic effect of bST in growing steers.

The IPCS Human Relevance Framework was evaluated for a DNA-reactive (genotoxic) carcinogen, 4-aminobiphenyl, based on a wealth of data in animals and humans. The mode of action involves metabolic activation by N-hydroxylation, followed by N-esterification leading to the formation of a reactive electrophile, which binds covalently to DNA, principally to deoxyguanosine, leading to an increased rate of DNA mutations and ultimately to the development of cancer. In humans and dogs, the urinary bladder urothelium is the target organ, whereas in mice it is the bladder and liver; in other species, other tissues can be involved. Differences in organ specificity are thought to be due to differences in metabolic activation versus inactivation. Based on qualitative and quantitative considerations, the mode of action is possible in humans. Other biological processes, such as toxicity and regenerative proliferation, can significantly influence the dose response of 4-aminobiphenyl-induced tumors. Based on the IPCS Human Relevance Framework, 4-aminobiphenyl would be predicted to be a carcinogen in humans, and this is corroborated by extensive epidemiologic evidence. The IPCA Human Relevance Framework is useful in evaluating DNA-reactive carcinogens.

The opportunity to write a historical review of the field of radiation biology allows for the viewing of the development and maturity of a field of study, thereby being able to provide the appropriate context for the earlier years of research and its findings. The pioneering work of Muller, Sax, and McClintock, and many others, has stood the test of time. The idea that x-rays could damage the genetic material and result in interactions that could lead to gene mutations and a range of chromosomal alterations is now interpretable in terms of induced DNA damage and errors of DNA repair. The expanded idea that such genetic alterations can be induced by DNA damage that is produced by one or two tracks of ionizing radiation remains the mainstay of radiation biology. The impact of the more recent molecular approaches to unraveling the mechanism behind this simple concept has confirmed this fundamental observation. The remarkable advances have allowed for a fairly complete understanding of the specific types of DNA damage induced by ionizing radiations and the pivotal role played by the errors of repair of double-strand breaks. Given our considerably enhanced knowledge of the details of the DNA repair processes involved, misrepair is a very unlikely event. The role of potential confounders of the concept of dose-response (e.g., bystander effects, genomic instability, and adaptive responses) is taking on a growing importance to the field. The evolving need is to begin to consider mechanistically-based dose-response models for cancer risk such that any potential impact of confounders on the response at low, environmental doses can be assessed. Thus, radiation biology research has always had a focus on how best to protect human health from radiation exposures and will continue to do so.

A cytosensor based on living chromatophores from Betta splendens Siamese fighting fish was used to test several classes of biologically active agents. Tested agents include neurotransmitters, adenyl cyclase activators, cytoskeleton effectors, cell membrane effectors and protein synthesis inhibitors. Characteristic cell responses were analyzed, and potential cytosensor applications were considered. Streptococcus pyogenes toxins streptolysin S and streptolysin O, Clostridium tetani tetanolysin, Staphylococcus aureus alpha-toxin and Vibrio parahemolyticus hemolysin, all bacterial toxins that act on cell membranes, elicited a strong response from chromatophores. A comparison of purified toxin to actual bacterial culture from Vibrio parahemolyticus demonstrated a nearly identical chromatophore cell response pattern. This suggests that the cytosensor response is reflective of bacterial toxin production.

The lacI mutant frequency and mutation spectrum were determined in the bone marrow and testes of B6C3F1 lacI transgenic mice exposed by inhalation to ethylene oxide (EO). Groups of male transgenic lacI B6C3F1 mice were exposed to 0, 25, 50, 100 or 200 p.p.m. EO for up to 48 weeks (6 h/day, 5 days/week) and were killed at 12, 24 or 48 weeks of EO exposure for determination of lacI mutant frequency. In the bone marrow, the lacI mutant frequency was significantly increased at the two highest exposure levels (100 and 200 p.p.m.) and at the 48 week exposure time point. The shape of the exposure-response curve for lacI mutant frequency in the bone marrow was non-linear. DNA sequence analysis of the bone marrow mutation spectrum revealed that only AT-->TA transversions occurred at an increased frequency in EO-exposed mice: 25.4% in EO-exposed mice for 48 weeks (200 p.p.m.) compared with 1.4% in air controls. In testes, the lacI mutant frequency was increased at a single exposure level of 200 p.p.m. for 24 weeks. At 48 weeks, the lacI mutant frequency in testes was significantly increased to an equal degree at 25, 50 and 100 p.p.m. EO but not at 200 p.p.m. Analysis of the testes mutation spectrum in air control mice and in mice exposed to 200 p.p.m. EO for 48 weeks revealed that no single mutational type occurred at an increased frequency. In the testes, there was a small increase across all mutational types that was sufficient to increase the overall lacI mutation frequency although not significant individually. The mutation spectrum in testes of EO-exposed mice also revealed that the increased lacI mutant frequency observed at 25 or 50 p.p.m. EO was not due to an increase in mutant siblings (clonality). These data demonstrate that inhalation exposure to EO for up to 48 weeks produces distinct mutagenic responses in bone marrow and testes.

Exposures to doses of radiation of 1-10 Gy, defined in this workshop as moderate-dose radiation, may occur during the course of radiation therapy or as the result of radiation accidents or nuclear/radiological terrorism alone or in conjunction with bioterrorism. The resulting radiation injuries would be due to a series of molecular, cellular, tissue and whole-animal processes. To address the status of research on these issues, a broad-based workshop was convened. The specific recommendations were: (1) RESEARCH: Identify the key molecular, cellular and tissue pathways that lead from the initial molecular lesions to immediate and delayed injury. The latter is a chronic progressive process for which postexposure treatment may be possible. (2) Technology: Develop high-throughput technology for studying gene, protein and other biochemical expression after radiation exposure, and cytogenetic markers of radiation exposure employing rapid and accurate techniques for analyzing multiple samples. (3) Treatment strategies: Identify additional biological targets and develop effective treatments for radiation injury. (4) Ensuring sufficient expertise: Recruit and train investigators from such fields as radiation biology, cancer biology, molecular biology, cellular biology and wound healing, and encourage collaboration on interdisciplinary research on the mechanisms and treatment of radiation injury. Communicate knowledge of the effects of radiation exposure to the general public and to investigators, policy makers and agencies involved in response to nuclear accidents/events and protection/treatment of the general public.

Chronic hypoxia caused by migration of native sea-level dwellers to high altitude or chronic lung disease leads to the development of increased pulmonary vascular resistance and pulmonary hypertension. This altitude-induced hypertension offers no obvious benefit and may indeed be maladaptive. A major mechanism thought to contribute to the development of pulmonary hypertension is hypoxia-induced loss of small blood vessels, sometimes termed rarefaction or pruning. More recent evidence caused us to question this widely accepted concept including the potent angiogenic effect of chronic hypoxia in all other vascular beds and the demonstration that new vessels can form in the pulmonary circulation when stimulated by chronic infection and lung resection. We tested the hypothesis that chronic environmental hypoxia causes angiogenesis in the adult pulmonary circulation by using stereological techniques combined with confocal microscopy to examine the resultant changes in pulmonary vascular structure in rats. We found that chronic hypoxia resulted in increased total pulmonary vessel length, volume, endothelial surface area and number of endothelial cells in vivo. This is the first reported demonstration of hypoxia-induced angiogenesis in the mature pulmonary circulation, a structural adaptation that may have important beneficial consequences for gas exchange. These findings imply that we must revise the widely accepted paradigm that hypoxia-induced loss of small vessels is a key structural change contributing to the development of pulmonary hypertension in high altitude adaptation and chronic lung disease.

Cancer risk assessment involves the steps of hazard identification, dose-response assessment, exposure assessment, and risk characterization. The rapid advances in the use of molecular biology approaches has had an impact on all 4 components, but the greatest overall current and future impact will be on the dose-response assessment because this requires an understanding of the mechanisms of carcinogenesis, both background and induced by environmental agents. In this regard, hazard identification is a qualitative assessment and dose-response is a quantitative estimate. Thus, the latter will ultimately require a quantitative assessment of molecular endpoints that are used to describe the dose-response for cancer. It has been possible for many years to quantitate alterations at the level of the single gene. For example, analysis of mutation frequency by phenotypic selection, analysis of transcription (mRNA) by Northern blot, analysis of translation (proteins) by Western blot, and analysis of kinetics of metabolism from metabolite levels. However, it is becoming clear that it is necessary when considering risk for adverse health outcomes to develop quantitative approaches for whole cell phenotypes or organ effects. For example, cancer is a whole tissue phenotype, not a feature of single gene mutations, in spite of the multistep (multimutation) mode of formation of a tumor. Thus, there is the need to quantitate the circuitry of a cell: the metabolic/biochemical pathways, genetic regulation pathways, and signaling pathway s in normal and stressed conditions. The hypothesis presented by Hanahan and Weinberg of the requirement for 6 acquired characteristics for tumor development, independent of tissue type and species or inducer, seems to provide a viable approach. This hypothesi s can be addressed through whole cell molecular assessment using microarray s and quantitative PCR together with the emerging proteomic approaches. This is the world of the new computational cell biology.

A two-cell biosensor was developed that uses optically detected changes in naturally colored fish chromatophores to measure the neurosecretory output of mammalian neuronal cells. The specific version of the biosensor described here is a continuous flow device that places red-pigmented, dendritic erythrophore cells directly downstream of an immobilized population of PC12 neuronal cells, a well-established model cell-line having neuroendocrine function. Agents known to stimulate catecholamine neurosecretion (secretagogues) were presented to the PC12 cells. It was found that the varying level of neurosecretion from the PC12 cells was measurable by judging the degree of pigment aggregation in the erythrophores. Increases in catecholamine secretion and consequent pigment aggregation were observed for several known secretagogues, including receptor agonists (ATP, acetylcholine), membrane depolarizing agents (high K+ concentration), and specific neurotoxins (black widow spider venom, α-latrotoxin). This particular two-cell biosensor, which is applicable to the detection of any agents that affect the levels of catecholamine secretion from PC12 cells, demonstrates the general principle that the breadth of sensitivity of a biosensor is increased by employing coupled cell types.

Previous studies have shown that p53 is involved in the repair of bleomycin-induced DNA damage, and that the frequency of bleomycin-induced chromatid aberrations is elevated in G(2)-treated p53 null transgenic mouse embryo fibroblasts (MEF) as compared to isogenic controls. To further characterize p53-mediated DNA repair, we studied the effect of p53 status on the ability of the DNA repair inhibitor 1-ss-D-arabinofuranosylcytosine (AraC) to sensitize MEF to bleomycin-induced chromatid aberrations. Both p53+/+ and p53-/- MEF were treated in G(2) with 0 to 7.5 microg/ml bleomycin in the presence or absence of AraC (5x10(-5) M). The frequency of bleomycin-induced chromatid aberrations was significantly higher in p53-/- cells than wild-type cells in the absence of AraC. AraC treatment significantly increased the frequency of bleomycin-induced chromatid aberrations in p53+/+ MEF to the levels in p53-/- (no AraC) but had no effect in p53-/- MEF. These results suggest that an AraC-sensitive DNA repair component is altered or absent in p53-/- cells. Similar results were observed in p53-mutant WTK1 and wild-type TK6 human lymphoblast cells exposed to 0 to 3 microg/ml bleomycin in G(2). However, AraC did cause a small increase in bleomycin sensitivity in WTK1 cells. This difference from the p53-/- MEF response may be due to differences in p53-mutant phenotype. To determine whether mutation of p53 alters DNA replication fidelity, p53+/+ and p53-/- MEF were exposed to 0 to 1 microg/ml mitomycin C (MMC). MMC did not induce chromosome aberrations in either cell line treated in G(2) but did with the same effectiveness in both cell lines treated in S-phase. Thus, p53 deficiency does not affect DNA replication fidelity or the repair of MMC-induced DNA damage.

The expression of the major porcine xenoantigens (Galalpha1-3Gal) in different tissues varies between species. The selection of suitable donors and the interpretation of studies which attempt to prevent hyperacute rejection are dependent on donor expression of Galalpha1-3Gal. Screening of large number of animals to find potential Galalpha1-3Gal negative donors requires a robust, tissue-based and practical method of assessing Galalpha1-3Gal expression. In this study, we have assessed the expression of Galalpha1-3Gal in a variety of pig organs using anti Galalpha1-3Gal antibody. Biopsies of heart, kidney, ear and tail were obtained from 20 outbred pigs. Biopsies were fixed in formalin and stained with a human anti Galalpha1-3Gal antibody obtained from pooled human AB serum passed down a Galalpha1-3Gal immunoadsorbent column. Tissue from all 4 organs from all 20 pigs expressed Galalpha13Gal. This study shows that detection of Galalpha1-3Gal on an ear or tail biopsy is a simple but very reliable method for assessing Galalpha1-3Gal expression on the heart and kidney and facilitates donor selection for xenotransplantation.

Cytogenetic assays are an integral component of the battery of short-term assays that are used for the hazard identification component of a cancer risk assessment. The protocol for the conduct of such assays for maximal sensitivity for detecting clastogenicity has to be attendant to the mechanism of induction of the endpoint being assessed and the fact that several aberration types are cell lethal necessitates that analysis be for cells at their first posttreatment metaphase. Cytogenetic assays for human populating monitoring have been used for predicting potential for carcinogenicity in humans. However, the assays as typically conducted are not appropriate for chronic exposures because nontransmissible alterations are assessed. The use of fluorescent in situ hybridization (FISH) techniques for the assessment of transmissible changes such as reciprocal translocations are required to make population monitoring studies interpretable, and for removing some of the concern over the influence of confounders on outcome. The database for the cytogenetic effects of ethylene oxide in vitro and in vivo, with an emphasis on human population monitoring, has been critically reviewed. Based on the endpoints studied, the size of the study groups, the information on exposure, the nature of any exposure response data, and the possible influence of confounders (i.e., control matching), it is concluded that acute, high exposures to ethylene oxide with sampling shortly (a few days) after exposure can be detected by increases in chromosome aberrations or SCE in peripheral lymphocytes. Such increases are indicators of exposure to a genotoxic chemical and not predictors of subsequent adverse health effects to individuals. The effect of chronic and/or low level (less than about 25 ppm) exposures cannot be reliably evaluated using current methods. The use of FISH, for example, for assessing reciprocal translocation frequencies (as a measure of transmissible events) will greatly improve the ability to detect chronic exposures to clastogenic chemicals.

The cytotoxic action of such alkylating chemotherapeutic drugs as 2-chloroethyl-N-nitrosourea (CNU) derivatives is countered by the repair protein O6-methylguanine-DNA methyltransferase (MGMT), which removes O6-alkylguanine induced in the DNA by these agents. Resistance to these drugs is often correlated with the MGMT levels in normal and tumor cells of human and rodent origin. Exposure of mouse 3T3 cells to increasing concentrations of CNU, and subsequent selection of resistant cells, led to the isolation of clones with 5-10 times higher levels of MGMT activity than in the control. The increased MGMT expression at both mRNA and protein levels resulted from 5- to 10-fold amplification of the Mgmt gene. Amplification of this gene was not associated with concomitant amplification of another alkylation damage repair gene, N-methylpurine-DNA glycosylase. No amplification of at least three other genes on chromosome 7 (which contains the Mgmt gene) was observed in the drug-resistant cells. Furthermore, the amplified Mgmt sequence was not associated with a homogeneously staining region, or double minute chromosomes, nor present as episomal DNA. In situ hybridization of metaphase chromosomes of the drug-resistant cells indicated both translocation and localized amplification of the Mgmt gene.

Telomeres in most species consist of repeat units of a small number of nucleotides that together with secondary structures and associated proteins stabilize the linear chromosomal DNA molecule. Chromosomes lose a small amount of telomeric DNA after each cell replication. It has been proposed that when telomeres shorten below a critical length, a DNA damage response pathway is activated and induces cell cycle arrest. In cells such as stem cells that maintain a proliferative capacity, telomere length is maintained by the reverse transcriptase, telomerase. In addition, telomerase activity is present in 90% of primary human tumors, suggesting a role for telomerase in providing a proliferative capacity to cells, which is a requirement in progression to malignancy. Telomerase activity can be involved in chromosome healing, although telomerase-independent processes also appear to be capable of capping broken chromosome ends. This review describes the structure and maintenance of telomeres, the importance of a critical telomere length to cell proliferation and the telomeric status of broken chromosome ends produced during development or by spontaneous or induced DNA damages.

Crossbred steers (n = 252, BW = 379 +/- 28 kg) were allotted to 42 pens in a 2 x 3 factorial arrangement of treatments: control or steroid implant (STR; estradiol benzoate+progesterone [three lighter blocks reimplanted on d 84] and trenbolone acetate [reimplanted on d 63]), and either 0, 80, or 160 mg/wk of recombinant bovine somatotropin (bST). Steers were adapted to the finishing diet (12% roughage equivalent, 13% CP) before the start of the experiment and fed for 84 or 119 d. Blood samples were taken on d 0, 14, 28, 56, and 84 for plasma urea N (PUN), serum somatotropin (ST), plasma insulin-like growth factor I (IGF-I), and plasma amino acid assay. Few interactions were noted (P.1). Gain was increased by both treatments: 1.30 vs 1.66 kg/d for control vs. STR (P.001) and 1.44, 1.49, and 1.51 kg/d (linear, P = .07) for 0, 80, and 160 mg of bST/wk, respectively. Gain efficiency was also improved: 169 vs 205 g/kg (P.001) and 177, 189, and 195 g/kg (linear, P.001), respectively. Average PUN was decreased (P.001) 29% by STR and decreased 17 and 29% by 80 and 160 mg of bST/wk, respectively (linear, P.001). Somatotropin decreased mean serum ST compared with controls; STR increased ST 36% compared with controls. Average plasma IGF-I was increased (P.001) 12% by STR and 13 and 19% (linear, P.001) by 80 and 160 mg of bST/wk, respectively. Both STR and bST influenced (P.05) plasma amino acid profiles. Indicators of carcass fatness were decreased linearly (P.05) by bST; STR implant tended to decrease carcass fatness and increase longissimus muscle area, which was related to carcass weight. The anabolic effects of STR and bST were found to be additive and possibly independent in feedlot steers.

Renal parenchymal disease is the most common cause of secondary hypertension, accounting for 2.5% to 5.0% of all cases. Hypertension associated with renal parenchymal disease occurs as a complication of a wide variety of glomerular and interstitial renal diseases and may accelerate the decline in renal function if inadequately controlled. Renal parenchymal hypertension most probably represents the combined interactions of multiple independent mechanisms: potential factors include impaired sodium handling leading to volume expansion, perturbations of the renin-angiotensin system, alterations in endogenous vasodepressor compounds, and possibly increased activity of vasoactive substances. The past several years have witnessed newer insights into both the pathophysiology and the therapeutics of this disorder. The characterization of endothelin and the nitric oxide (NO)-arginine pathway and their roles in biology and medicine has provided additional new insights with regard to the pathogenesis of hypertension in renal parenchymal disease. For example, methylated L-arginine derivatives that possess NO synthase inhibitor capabilities including NG-N-dimethylarginine and N-monomethyl-L-arginine are found in human plasma and in urine. Patients with chronic uremia have impaired elimination of these compounds, and circulating concentrations of these compounds may increase sufficiently to result in inhibition of NO production. Thus, accumulation of endogenous NO synthase inhibitors might contribute to the hypertension of advanced renal failure. Similarly, it has been proposed that increased endothelium-derived endothelin that results from hypertensive injury to vascular endothelium could lead to further vasoconstriction and worsening of hypertension. Additional insight into this fascinating problem must await further biochemical characterization of some of the mediators and a more precise delineation of their pathophysiological role.

The effects of dietary virginiamycin level on performance and liver abscesses in feedlot cattle were evaluated in seven dose-response studies. Steers and heifers were fed finishing diets ranging in energy content from 1.34 to 1.51 Mcal of NEg/kg of DM. In all studies, virginiamycin added to the diet improved average daily gain and(or) feed conversion, with no substantial effect on dry matter intake. Pooled analyses of four studies providing virginiamycin at 11.0, 19.3, and 27.6 mg/kg of DM in the complete diet indicated that growth and feed conversion were linearly improved (P < .05); feeding 19.3 mg/kg improved these measurements by 3.0 and 3.8%, respectively. Overall incidence (score 0 vs score 1, 2, and 3) and severity (score 0, 1, and 2 vs score 3) of liver abscesses were reduced (P < .01) by feeding virginiamycin at either 19.3 or 27.6 mg/kg. Linear plateau modeling indicated that the effective dose range for virginiamycin in feedlot diets (DM basis) was 19.3 to 27.3 mg/kg for increasing average daily gain, 13.2 to 19.3 mg/kg for improving feed conversion, and 16.5 to 19.3 mg/kg for reducing liver abscess incidence.

Results from new chromosome studies in laboratory animals, comparative investigations of styrene metabolism and pharmacokinetics in humans and animals, and several recent cytogenetic surveys of styrene-exposed workers have necessitated a comprehensive re-evaluation of the chromosome-damaging effects of this chemical. Both styrene and its genotoxic metabolite, styrene oxide, can induce chromosome aberrations (CA) and sister chromatid exchanges (SCE) in vitro, but the chromosome-damaging ability of styrene is only manifested if test conditions favour its metabolic activation over inactivation. There is no convincing evidence of styrene clastogenicity in experimental animals. Styrene oxide is clastogenic only at lethal concentrations via i.p. injection in Chinese hamsters (but not via inhalation) or after oral treatment of mice, a route considered inappropriate for investigating the chromosome-damaging potential of inhaled styrene in man. Styrene and styrene oxide can induce SCE in animals at very high concentrations. Eighteen of 52 cytogenetic studies (CA, micronuclei, SCE) on peripheral blood lymphocytes of styrene workers have reported increases in chromosome damage. The positive findings are not compatible with the conclusion that styrene is responsible for the cytogenetic effects for the following reasons. 1. (a) The positive or negative outcome of the various investigations bears no relationship to the degree of exposure of the workers. 2. (b) There is no convincing evidence of a positive dose response relationship. 3. (c) The relative induction of CA and SCE in worker studies are the opposite of observations of styrene effects in cultured lymphocytes and in laboratory animals. 4. (d) The reports of chromosome-type exchanges in some studies of styrene workers is inconsistent with observations of styrene clastogenicity in cultured lymphocytes. 5. (e) Reports of SCE induction in workers exposed to low concentrations of styrene are not compatible with results of animal inhalation studies, particularly in view of the differences in styrene metabolism and pharmacokinetics between humans and rodents. The increases in cytogenetic effects reported in some studies on styrene workers are probably attributable to the presence of other chromosome-damaging agents in the workplace and/or to inadequate investigations.

The celebration of the 25th Anniversary of the Environmental Mutagen Society provides an excellent opportunity to assess the status of research in a broad range of areas, with an emphasis on the directions in which they are going. This chapter concentrates on the analysis of chromosomal alterations in mammalian germ cells. The future developments in germ cell cytogenetics research will build heavily upon techniques developed over the past 25 years. With these it is possible to assess numerical and structural alterations in the male in differentiating spermatogonia, spermatocytes, and post-mei-otic cells (at the first cleavage division) and for the female in oocytes and the zygote. The most predictable advances will be in the identification of specific alterations through FISH of interphase spermatozoa in humans and further improvements with the human sperm/hamster egg in vitro fertilization technique. Of particular importance is the fact that this will allow for the study of effects in human germ cells. From a more speculative viewpoint it might be possible to assess the role of particular genomic organization on genetic outcomes by direct observation; these might include genomic imprinting and the visual separation of male and female genomes. The overall aim of germ cell cytogenetic studies will remain as improving our ability to identify and estimate the true genetic risk in humans. © 1994 Wiley-Liss, Inc.

A large number of studies have been reported on the genotoxicity of styrene in vitro and in vivo and the potential effects on humans of occupational exposure. Because of a variety of technical problems and difficulties in data interpretation, it has not been clearly established whether styrene can induce chromosomal aberrations and/or sister chromatid exchange (SCE) in vivo in animals or humans. The importance of clarifying this situation led to the development of the study described in this paper. Male Fischer 344 rats were exposed to styrene at concentrations of 150, 500 or 1000 ppm for 6 h/day on 5 days/week for 4 weeks. A negative control (air) was included. An additional control (ethylene oxide, 150 ppm) group was included in an attempt to establish the usefulness of rat lymphocytes for cytogenetic analysis in this protocol of long-term exposure by inhalation. The choice of agent and of exposure was based on the expectation that they would produce a positive response for SCE and/or chromosomal aberrations under the assay conditions used. Peripheral blood samples were drawn at 1, 2, 3 and 4 weeks of exposure and at 4 weeks after the end of exposure. Cultures were established, and SCE (second mitosis) and chromosomal aberrations (first mitosis) were analysed. The frequency of chromosomal aberrations was not increased over that in the air controls in the animals exposed to styrene or ethylene oxide at any of the sampling times. Styrene did not induce SCE at any of the concentrations or sampling times; however, the frequency of SCE was increased following exposure to ethylene oxide at all sampling times, with a positive exposure-response relationship with time of exposure as the variable. The data are compared with other, similar sets reported in the literature, and their significance for predicting responses in people occupationally exposed to styrene is discussed.

Two experiments were conducted at three locations to determine the correct dosage and carrier for trenbolone acetate (TBA) and estradiol (E2) implants in feedlot steers. In the dose-response experiment, 1,296 steers were allotted to six implant treatments (48 pens per location): control, 140 mg of TBA (140/0), 30 mg of E2 (0/30), 20 mg of TBA + 4 mg of E2(20/4), 80 mg of TBA + 16 mg of E2(80/16), and 140 mg of TBA + 28 mg of E2 (140/28). In the carrier experiment, 575 steers were allotted to five implant treatments (25 pens per location): control, 140 mg of TBA + 28 mg of E2 in lactose (140/28-LA), 140 mg of TBA + 28 mg of E2 in cholesterol (140/28-CH), 140 mg of TBA + 20 mg of E2 in LA (140/20-LA), and 200 mg of progesterone + 20 mg of E2 benzoate (SS, reimplanted). In both experiments steers were fed a finishing diet for 140 to 168 d. In the dose-response experiment, response to TBA alone (140/0) did not differ from control (P greater than .2). Estradiol alone (0/30) improved ADG by 7% (P less than .01) and tended to improve feed efficiency over control (3%, P = .17). The highest dosage (140/28) improved ADG by 18% (P less than .001) and feed efficiency by 10% (P less than .001) over control and 10% (P less than .001) and 7% (P less than .01) over E2 alone, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Two studies were conducted to determine whether sodium thiosulfate (THS) can estimate extracellular water (ECW) in beef cattle in conjunction with empty body water (EBW) estimation by urea space. Experiment 1 used 24 steers (366 kg) to determine the clearance parameters for THS and urea. Blood samples were taken over 1 h. A two-component curve, Y = A1ek1(t) + A2ek2(t), (t = hours after infusion) fit the clearance of both markers; intercepts (A1, A2) and clearance coefficients (k1, k2) were 44.8, 44.4, -25.8, and -2.24 mg/dL, respectively, for THS (r2 = .98, Sy.x = 2.72, animal effects removed and 24.4, 10.5, -21.7, and -.71 mg/dL, respectively, for urea (r2 = .98, Sy.x = 1.49). Sodium thiosulfate equilibrated with ECW 5 to 10 min after infusion. Experiment 2 consisted of 22 steers (483 kg) infused with a combination solution of 20% urea, 10% THS, and 4% sodium thiocyanate (SCN; equilibration time = 28 min); half the steers were implanted with estradiol. Empty body water increased with implantation (P less than .01). Extracellular water tended to increase in implanted steers as measured by THS (12 min, P = .14) and SCN (P = .10). The estimation of ECW at 12 min was not different (P greater than .2) from the SCN estimate at 28 min (SCN = 3.7 + .873 THS; r2 = .70; P less than .001). Sodium thiosulfate gave reasonable estimates of ECW (22 to 26% of BW) and required only 0- and 12-min blood samples.(ABSTRACT TRUNCATED AT 250 WORDS)

In order to study the mechanism of induction of mutations and chromosome aberrations by ionizing radiations, it is particularly useful to have available radiation-sensitive mutants. While several X-ray-sensitive rodent cell lines are available, they have been selected rather nonspecifically. It was determined that selection for resistance to the DNA replication/repair inhibitor, 1-beta-D-arabinofuranosylcytosine (ara-C), would permit production of a set of X-ray-sensitive mutant cell lines that would be defective in the resynthesis step of excision or recombination repair. Such mutant cells could also be used for the isolation and characterization of human DNA repair genes. In particular, it was predicted that the repair gene defective in individuals with ataxia telangiectasia (AT) might be amenable to study with ara-C-resistant (X-ray-sensitive) mutants, since additional studies, presented here, have shown that AT cells are resistant to ara-C. In the long term, it is hoped that determining the specific defect in AT might lead to an understanding of the possible role of defective repair in tumor induction and/or progression. The general approach used to isolate ara-C-resistant Chinese hamster ovary cell mutants was to treat cells with ethyl methanesulfonate and select in increasing concentrations of ara-C. Although several mutants were isolated, one in particular, Ara-CR213, has been studied most extensively. It was selected largely because it shows the greatest sensitivity to X-rays. Ara-CR213 cells were hypersensitive to the killing effect of X-rays with an LD10 of 2.5 Gy as compared to the wild-type cells that had an LD10 of 6 Gy. The mutant showed an increased frequency of X-ray-induced chromosomal aberrations in the G1 and G2 stages of the cell cycle compared to wild-type frequencies. There was no increase in sister chromatid exchange levels. All of these observations in Ara-CR213 are very similar to those made with AT cells in our and other laboratories. Even more important, complementation analysis of Ara-CR213 x AT hybrid cells indicated that the gene responsible for X-ray sensitivity of AT is also mutated in Ara-CR213 cells. Thus, Ara-CR213 appears to have a mutant phenotype and probably genotype that is very similar to, if not exactly the same as, those of AT. This makes it quite different from other X-ray-sensitive cells that have been isolated in other laboratories.

Because roughage in feedlot diets is one of the most expensive ingredients on an energy basis, regimens that minimize roughage usage are of interest. Crossbred steers of British breeds (n = 112, initial BW = 405 kg) were used to compare the feeding of diets containing 2% roughage from d 22 through 84 and 10% roughage from d 85 to finish (d 133; 2/10%) to the feeding of 10% roughage throughout the finishing period (10/10%); all diets were based on steam-flaked sorghum grain and contained monensin and tylosin. When the 2% roughage diet was fed, steers consumed less feed (6.8 vs 7.8 kg/d, P less than .01), tended to gain less (1.11 vs 1.20 kg/d, P = .13), and were numerically more efficient (16.5 vs 15.5 kg of gain/100 kg of DMI, P greater than .2) than steers fed the 10% roughage diet (10/10%). After the roughage content was increased from 2 to 10% on d 85 (all steers fed 10% roughage), steers fed the 2/10% regimen had greater DMI (8.4 vs 8.0 kg/d, P = .08) and ADG (1.29 vs 1.09 kg, P = .06), and tended to be more efficient (15.4 vs 13.6 kg of gain/100 kg of DMI, P = .10) than steers fed the 10/10% regimen. Steers fed the two regimens had similar (P greater than .2) overall gain performance. The 2/10% regimen tended to have a greater percentage of Choice carcasses (58 vs 42%, P = .14) and numerically more liver abscesses (24 vs 15%, P greater than .2) than the 10/10% regimen.(ABSTRACT TRUNCATED AT 250 WORDS)

Chinese hamster ovary cells with no detectable (less than 200 molecules/cell) O6-methylguanine-DNA methyltransferase (EC 2.1.1.63) were transfected with human cell DNA and pSV2neo plasmid by electroporation. Two stable transformant clones, GC-1 and GC-2, containing 4 X 10(4) and 4-6 X 10(3) methyltransferase molecules/cell respectively were isolated by successive screening in the presence of G418 and 2-chloroethyl-N-nitrosourea (CNU). Only three or four copies of pSV2neo DNA and no repetitive human DNA sequence were detected in these isolates. Secondary transfection of parent cells with GC-1 DNA yielded several clones containing 2-10 X 10(3) methyltransferase molecules/cell. The rate of removal of O6-methylguanine in GC-1, GC-2 and parent cells in vivo reflected their methyltransferase levels, while the N-methylpurines were removed at similar rates in all three cell lines. The differential sensitivity of these cells to several alkylating agents, namely CNU, N-methyl-N-nitrosourea, N-methyl-N'-nitro-N-nitrosoguanidine and methyl-methane sulfonate (MMS), known to yield different proportions of O6-alkylguanine among the alkyl adducts in DNA, varied widely. The largest and smallest differences in toxic response were observed with CNU and MMS respectively. These cell lines showed no difference in sensitivity to the DNA cross-linking agent psoralen. These data strongly suggest that alkylating agents produce two classes of lethal lesions, one of which is O6-alkylguanine. Induction of mutations at the hypoxanthine-phosphoribosyltransferase locus in these cells lines suggests that, regardless of its relative yield, O6-methylguanine is the major mutagenic lesion for all alkylating agents.