The gastrointestinal syndrome and mucosal clonogenic cells: relationships between target cell sensitivities, LD50 and cell survival, and their modification by antibiotics

The sensitivity of the target cells responsible for the gastrointestinal syndrome in mice was deduced from the steepness of the dose-survival curve for mice assessed on Day 7 after irradiation. The D0 value was 1.25 +/- 0.22 Gy, virtually identical to the value of 1.23 +/- 0.08 measured for microcolony-forming cells (clonogens) over about the same range of dose in concurrent experiments. The survival of clonogens was similar when assayed in mice surviving to Days 3, 4, or 5, but clonogenic sensitivity was lower when assessed on Day 7. This was shown at one dose to be due largely to a selection of mice with high colony counts with only a small contribution from crypt budding. The LD50 for mice corresponded to a surviving fraction of crypts of about 0.35. An injection of 5 mg streptomycin sulphate ip daily for 5 days after irradiation increased the latent period by about 1 day, increased the LD50 by about 1.4 Gy, but did not significantly change the survival of clonogens. These studies are the first to test and satisfy the interpretation of a dose-response curve for animal survival in terms of "target cell" survival, where measurements of both are made over a similar range of dose in concurrent experiments.     

DESIGN: computerized optimization of experimental design for estimating Kd and Bmax in ligand binding experiments. II. Simultaneous analysis of homologous and heterologous competition curves and analysis blocking and of "multiligand" dose-response surfaces

We have developed a computer program, DESIGN, for optimization of ligand binding experiments to minimize the "average" uncertainty in all unknown parameters. An earlier report [G. E. Rovati, D. Rodbard, and P. J. Munson (1988) Anal. Biochem. 174, 636-649] described the application of this program to experiments involving a single homologous or heterologous dose-response curve. We now present several advanced features of the program DESIGN, including simultaneous optimization of two or more binding competition curves optimization of a "multiligand" experiment. Multiligand designs are those which use combinations of two (or more) ligands in each reaction tube. Such designs are an important and natural extension of the popular method of "blocking experiments" where an additional ligand is used to suppress one or more classes of sites. Extending the idea of a dose-response curve, the most general multiligand design would result in a "dose-response surface". One can now optimize the design not only for a single binding curve, but also for families of curves and for binding surfaces. The examples presented in this report further demonstrate the power and utility of the program DESIGN and the nature of D-optimal designs in the context of more complex binding experiments. We illustrate D-optimal designs involving one radioligand and two unlabeled ligands; we consider one example of homogeneous and several examples of heterogeneous binding sites. Further, to demonstrate the virtues of the dose-response surface experiment, we have compared the optimal surface design to the equivalent design restricted to traditional dose-response curves. The use of DESIGN in conjunction with multiligand experiments can improve the efficiency of estimation of the binding parameters, potentially resulting in reduction of the number of observations needed to obtain a desired degree of precision in representative cases.     

Alternative methods for the median lethal dose (LD(50)) test: the up-and-down procedure for acute oral toxicity

The authors have developed an improved version of the up-and-down procedure (UDP) as one of the replacements for the traditional acute oral toxicity test formerly used by the Organisation for Economic Co-operation and Development member nations to characterize industrial chemicals, pesticides, and their mixtures. This method improves the performance of acute testing for applications that use the median lethal dose (classic LD50) test while achieving significant reductions in animal use. It uses sequential dosing, together with sophisticated computer-assisted computational methods during the execution and calculation phases of the test. Staircase design, a form of sequential test design, can be applied to acute toxicity testing with its binary experimental endpoints (yes/no outcomes). The improved UDP provides a point estimate of the LD50 and approximate confidence intervals in addition to observed toxic signs for the substance tested. It does not provide information about the dose-response curve. Computer simulation was used to test performance of the UDP without the need for additional laboratory validation.     

Dose-response curve estimation: a semiparametric mixture approach

In the estimation of a dose-response curve, parametric models are straightforward and efficient but subject to model misspecifications; nonparametric methods are robust but less efficient. As a compromise, we propose a semiparametric approach that combines the advantages of parametric and nonparametric curve estimates. In a mixture form, our estimator takes a weighted average of the parametric and nonparametric curve estimates, in which a higher weight is assigned to the estimate with a better model fit. When the parametric model assumption holds, the semiparametric curve estimate converges to the parametric estimate and thus achieves high efficiency; when the parametric model is misspecified, the semiparametric estimate converges to the nonparametric estimate and remains consistent. We also consider an adaptive weighting scheme to allow the weight to vary according to the local fit of the models. We conduct extensive simulation studies to investigate the performance of the proposed methods and illustrate them with two real examples.     

Optimal designs for a multiresponse Emax model and efficient parameter estimation

The aim of dose finding studies is sometimes to estimate parameters in a fitted model. The precision of the parameter estimates should be as high as possible. This can be obtained by increasing the number of subjects in the study, N, choosing a good and efficient estimation approach, and by designing the dose finding study in an optimal way. Increasing the number of subjects is not always feasible because of increasing cost, time limitations, etc. In this paper, we assume fixed N and consider estimation approaches and study designs for multiresponse dose finding studies. We work with diabetes dose–response data and compare a system estimation approach that fits a multiresponse Emax model to the data to equation‐by‐equation estimation that fits uniresponse Emax models to the data. We then derive some optimal designs for estimating the parameters in the multi‐ and uniresponse Emax model and study the efficiency of these designs.     

LOGIT: a program for dose-response analysis.

We describe a FORTRAN computer program for fitting the logistic distribution function: (formula: see text) Where x represents dose or time, to dose-response data. The program determines both weighted least squares and maximum likelihood estimates for the parameters alpha and beta. It also calculates the standard errors of alpha and beta under both estimation methods, as well as the median lethal dose (LD50) and its standard error. Dose--response curves found by both fitting methods can be plotted as well as the 95% confidence bands for these lines.     

Maximum likelihood estimation for cytogenetic dose-response curves

In vitro dose-response curves are used to describe the relation between chromosome aberrations and radiation dose for human lymphocytes. The lymphocytes are exposed to low-LET radiation, and the resulting dicentric chromosome aberrations follow the Poisson distribution. The expected yield depends on both the magnitude and the temporal distribution of the dose. A general dose-response model that describes this relation has been presented by Kellerer and Rossi (1972, Current Topics on Radiation Research Quarterly 8, 85-158; 1978, Radiation Research 75, 471-488) using the theory of dual radiation action. Two special cases of practical interest are split-dose and continuous exposure experiments, and the resulting dose-time-response models are intrinsically nonlinear in the parameters. A general-purpose maximum likelihood estimation procedure is described, and estimation for the nonlinear models is illustrated with numerical examples from both experimental designs. Poisson regression analysis is used for estimation, hypothesis testing, and regression diagnostics. Results are discussed in the context of exposure assessment procedures for both acute and chronic human radiation exposure.     

Design and analysis of phase I clinical trials

The Phase I clinical trial is a study intended to estimate the so-called maximum tolerable dose (MTD) of a new drug. Although there exists more or less a standard type of design for such trials, its development has been largely ad hoc. As usually implemented, the trial design has no intrinsic property that provides a generally satisfactory basis for estimation of the MTD. In this paper, the standard design and several simple alternatives are compared with regard to the conservativeness of the design and with regard to point and interval estimation of an MTD (33rd percentile) with small sample sizes. Using a Markov chain representation, we found several designs to be nearly as conservative as the standard design in terms of the proportion of patients entered at higher dose levels. In Monte Carlo simulations, two two-stage designs are found to provide reduced bias in maximum likelihood estimation of the MTD in less than ideal dose-response settings. Of the three methods considered for determining confidence intervals--the delta method, a method based on Fieller's theorem, and a likelihood ratio method--none was able to provide both usefully narrow intervals and coverage probabilities close to nominal.     

Pathogenicity and virulence

Invertebrate pathologists have multiple definitions for the terms pathogenicity and virulence, and these definitions vary across disciplines that focus on host-pathogen interactions. We surveyed various literatures, including plant pathology, invertebrate pathology, evolutionary biology, and medicine, and found most define pathogenicity as the broader term, which incorporates virulence. Virulence is seen as the severity of disease manifestation that can only be measured in infected individuals. These definitions readily apply to both lethal and non-lethal diseases. Invertebrate pathologists commonly use dose-response bioassays to estimate LD(50) or LC(50) (dose or concentration needed to kill 50% of hosts exposed). These bioassays measure pathogenicity if the bioassay includes a transmission component, and measure virulence if the bioassay is measured in infected individuals only. Another common bioassay estimate is LT(50) (median time to death of infected hosts), which is a measure of virulence as long as survivors are not included in its calculation.     

Insecticide susceptibility of Nezara viridula (Heteroptera: Pentatomidae) and three other stink bug species composing a soybean pest complex in Japan

The susceptibility of the stink bug species Nezara viridula (L.), Nezara antennata Scott, Piezodorus hybneri (Gmelin), and Riptortus pedestris (F.) to insecticides was tested, establishing their 50% lethal dose (LD50) values as baseline data. Third instars and adults of the four species were treated by topical application with seven insecticides: fenitrothion, fenthion, etofenprox, silafluofen, dinotefuran, clothianidin, and ethiprole. The weight of the stink bug and weight of the insecticide applied to each bug were used as explanatory variables in the probit regression analysis. The effect of the body weight on the dose-response relationship, the proportional model, was not uniform among the tested insecticide-stink bug combinations. However, the basic model fit all combinations and could estimate LD50 values successfully. Therefore, LD50 values at the medium (average) weight estimated by the basic model were selected to describe the susceptibility of the stink bugs. The LD50 value of silafluofen for N. viridula adults, and that of silafluofen and etofenprox for N. antennata adults, was at least 2,338 ng greater than the other species exposed to each insecticide. Almost all of the LD50 values for adults were over 10 times greater than those of the same species' nymphs treated with the same insecticide. Thus monitoring of occurring species and their developmental stages is important for controlling effectively the stink bug pest complex by insecticides, especially by silafluofen or etofenprox. The estimated LD50 values can be used as baseline data to compare the susceptibility of the species collected in another year or location.     

Interval estimation of the median lethal dose

Assuming a logistic dose-response curve, one can construct a confidence interval for the LD50 from the asymptotic likelihood ratio test. Reasons are given for preferring this likelihood ratio interval to the established interval calculated by applying Fieller's theorem to the maximum likelihood estimates.     

Independent effect of a mixed-beam regimen of fast neutrons and gamma rays on a murine fibrosarcoma

Biological effectiveness of a mixed-beam regimen of fast neutrons and photons was studied in an animal tumor system. NFSa , a spontaneous fibrosarcoma in a C3H mouse, was transplanted in the right hind legs of syngeneic male mice and locally irradiated with a single dose or five daily doses. Tumor control experiments showed that five gamma-ray doses increased TCD50 values by 20 Gy and produced a shallower slope on the dose-response curve compared to that after a single fraction. Fractionated neutron doses also increased the TCD50 value by 9 Gy without changing the slope of the dose-response curve. A mixed-beam regimen of N-gamma-gamma-gamma-N resulted in an independent effect on the tumor. Second, tumor cell survival was examined by the lung colony assay. Nembutal anesthesia reduced the tumor oxic cell fraction, resulting in a single component dose-response curve after a single gamma ray. Five fractionated doses of gamma rays increased both D0 and extrapolation number while those of fast neutrons increased only extrapolation number. The D0 and extrapolation number of the mixed-beam regimen were again identical to those values assuming that the mixed-beam effect was independent. RBEs obtained from cell survival were fairly close to those from TCD50 assays except single-dose experiments.     

Comparison of cytogenic response of human lymphocytes to in vivo and in vitro exposure to low doses of gamma-rays. Unstable chromosomal exchanges detected by FPG techniques

On peripheral lymphocytes of eight cancer patients undergone whole-body therapeutic irradiation (at daily dose of 10 cGy up to total dose of 50 cGy of 60Co gamma-rays) the dose-response of unstable chromosome exchanges (dicentrics and centric rings) was studied. This dose response fitted well linear function. The lower slope of dose-response curve was found for in vivo irradiated lymphocytes as compared to the dose response curve obtained for in vitro irradiated lymphocytes of the same patients. This finding seems to provide evidence that in case of protracted irradiation of individuals an absorbed dose could be underestimated if for biological dosimetry an in vitro dose response curve for unstable chromosome aberrations is used as referent one.     

Estimating LD50s without a computer

Quantitative analysis of dose-related effects, such as mosquitoes killed by insecticide or parasites killed by a drug, usually involves estimating the dose which kills, on average, 50% of the subjects. This quantity is often termed the LD(50) (LD for lethal dose), or the ED(50) (ED for effective dose). Other specified response levels, such as the LD(90) - the dose that kills 90% of subjects - may also be derived. Dose-related effects of this type follow an S-shaped curve because, clearly, doses lower than those giving zero response will also give zero response, while at the other end of the curve, doses above those giving a maximum response can also only give a maximum response. In other words the curve flattens out at both ends. The mathematics of fitting a suitable S-shaped curve to such data - for example by probit analysis - is quite simple in principle but can be arduous and time-consuming without a suitably programmed computer. In this article, Michael Healy explains an alternative approach which is particularly applicable to field observations where computers are unavailable.     

Uncertainties in the risk assessment of three mycotoxins: aflatoxin, ochratoxin, and zearalenone

The risk assessment of mycotoxins is made up of two major components: an exposure assessment and a hazard assessment. There are many uncertainties in both of these components. This paper will briefly discuss the various aspects of the risk assessment process as it applies to mycotoxins and will then focus mainly on some of the uncertainties in the hazard assessment component of several carcinogenic mycotoxins. To arrive at an estimated "safe dose" (end point of the hazard assessment), we have previously used two major approaches: the no observed effect level (NOEL) divided by a safety factor approach and a mathematical (robust linear) extrapolation to a "virtual safe dose." Both of these approaches use only points from the no observed effect region of the dose-response curve and ignore valuable data from the response region. It is proposed to use the dose at which 50% of the animals would have developed tumors (the TD50) divided by a large safety factor of 50,000 as an additional estimate of "safe dose". For many studies, the TD50 lies within the observed response region of the dose-response curve and may have more validity. It is also suggested in certain cases that some of the uncertainties regarding the NOEL can be reduced if one uses a statistically derived no effect level (NEL).     

Microwave Facilitation of Methylnaltrexone Antagonism of Morphine-Induced Analgesia in Mice

Exposure to low dose microwave irradiation has been postulated to influence effects of centrally active pharmacological agents. In this investigation, the analgesic dose, 50% (ADSO) for morphine in the mouse tail flick test was significantly increased by pretreatment with the centrally active opiate receptor blocker naltrexone, and the dose-response curve ms shifted significantly to the right. On the other hand, the AD50 and dose-response curve for morphine were not influenced by either the peripherally active opiate receptor blocker methylnaltrexone alone or microwave irradiation (2.45 GHz, 20 mW/cm², CW, 10 min) alone. However, following both methylnaltrexone pretreatment and microwave exposure, there was both a significant increase in the AD50 as well as a significant rightward shift of the dose-response curve, indicating a competitive antagonism of morphine by methylnaltrexone. One possible explanation for this microwave-drug interaction is that microwave energy might facilitate entry of methylnaltrexone into the central nervous system.     

Effects of ultraviolet radiation on carotenoid-containing and albino strains of Neurospora crassa

To determine the influence of carotenoids on UV sensitivity of Neurospora crassa conidia, the dose-response curves of 3 albino strains were compared to that of the wild type. In a control experiment the genetic background effects were eliminated by irradiating the wild type in which carotenoid synthesis had been inhibited by β-ionone, and comparing the dose-response curve to that of untreated wild type. All strains used had similar γ-ray survival curves and nuclei per conidium which precluded differences in UV sensitivity due to morphological differences.The albinos and β-ionone-treated strains showed a greater sensitivity (mean LD50 4.35·10³ erg/mm²) to UV irradiation than the wild type (LD50 7.30·10³ erg/mm²).     

The design of in vivo multifraction experiments to estimate the alpha-beta ratio

Using statistical methods, the designs of multifraction experiments which are likely to give the most precise estimate of the alpha-beta ratio in the linear-quadratic model are investigated. The aim of the investigation is to try to understand what features of an experimental design make it efficient for estimating alpha/beta rather than to recommend a specific design. A plot of the design on an nd2 versus nd graph is suggested, and this graph is called the design plot. The best designs are those which have a large spread in the isoeffect direction in the design plot, which means that a wide range of doses per fraction should be used. For binary response assays, designs with expected response probabilities near to 0.5 are most efficient. Furthermore, dose points with expected response probabilities outside the range 0.1 to 0.9 contribute negligibly to the efficiency with which alpha/beta can be estimated. For "top-up" experiments, the best designs are those which replace as small a portion as possible of the full experiment with the top-up scheme. In addition, from a statistical viewpoint, it makes no difference whether a single large top-up dose or several smaller top-up doses are used; however, other considerations suggest that two or more top-up doses may be preferable. The practical realities of designing experiments as well as the somewhat idealized statistical considerations are discussed.     

Differences in the acute intestinal syndrome after partial and total abdominal irradiation in mice

The acute intestinal syndrome in mice was analysed after partial (PAI) and total abdominal irradiation (TAI). The LD50/15 was significantly higher after PAI (16.3 Gy) than after TAI (14.3 Gy). The dose-response curve for maximal weight loss also showed a shift of 1.8-2 Gy to higher doses after PAI compared with TAI. The X-ray survival curve for duodenal crypt cells was shifted by only 0.6 Gy for PAI and TAI. In order to assess the possible role of radiation-induced leucopenia and the influence of irradiating the spleen (shielded with PAI), lethality, weight loss and blood leucocyte counts were compared after PAI and TAI in splenectomized and non-splenectomized mice. No major difference in leucopenia was found between the different treatment groups, whereas the differences in lethality and weight loss between PAI and TAI remained the same. Shielding the spleen in the partial abdominal field therefore did not contribute to the difference in LD50/15. These findings imply that the increased LD50/15 after PAI compared with TAI was mainly due to shielding of a small part of the bowel (about 13 per cent of the abdominal area).     

Comparison of dose-response relationships for ethyl methanesulfonate and 1-ethyl-1-nitrosourea in Drosophila melanogaster spermatozoa

The relative importance of different sites of alkylation on DNA was determined by comparing two ethylating agents. 1-Ethyl-1-nitrosourea (ENU) ethylates DNA with a higher proportion of total adducts on ring oxygens than ethyl methanesulfonate, which ethylates with a higher proportion of total adducts on the N-7 of guanine. Research with somatic cells in culture and prokaryotes strongly suggests that O6-guanine (O6-G) is the principal genotoxic site. To determine the importance in germ-line mutagenesis of the O6-G site relative to the N-7 of guanine, dose-response curves were constructed for both ENU and EMS, where dose was measured as total adducts per deoxynucleotide (APdN) and response as sex-linked recessive lethals (SLRL) induced in Drosophila melanogaster spermatozoa. For both mutagens the dose response curve was linear and extrapolated to the origin. The dose-response curve for ENU was fit to an equation m = 6.2D, and the dose response curve for EMS, from this and previous experiments, was m = 3.2D where m = %SLRL and D = APdN X 10(-3). Therefore, ENU is 1.9 times more efficient per adduct in inducing SLRL mutations than EMS. In vitro studies showed that ENU induced 9.5% of its total adducts on O6-G while EMS induced 2.0% of its adducts on O6-G. If O6-G was the sole genotoxic site, then ENU should be 4.8 times more efficient per adduct than EMS. In contrast, if N-7 G was the sole genotoxic site, ENU would be only 0.19 as effective as EMS. It was concluded that while O6-G was the principal genotoxic site, N-7 G made a significant contribution to germ-line mutagenesis.