One‐sided Simultaneous Confidence Intervals for Effective Dose Steps in Unbalanced Designs

An important issue in dose finding is whether a further dose increment leads to a relevant increase in efficacy. Clinical efficacy should not be considered by point zero null hypotheses. Instead, shifted hypotheses for the difference or the ratio can be used. Because the a priori definition of a relevance threshold is frequently difficult, confidence intervals should be used for a posteriori interpretation. Sample size estimation – a‐priori or by adaptive interim analysis‐ is inherent, because the effective dose steps are arbitrary in un‐designed studies. For simultaneous confidence intervals without order restriction the exact distributions under the null and the alternative hypothesis is proposed for the general unbalanced one‐way design.     

DNA damage responses at low radiation doses

Increased cell killing after exposure to low acute doses of X rays (0-0.5 Gy) has been demonstrated in cells of a number of human tumor cell lines. The mechanisms underlying this effect have been assumed to be related to a threshold dose above which DNA repair efficiency or fidelity increases. We have used cells of two radioresistant human tumor cell lines, one that shows increased sensitivity to low radiation doses (T98G) and one that does not (U373), to investigate the DNA damage response at low doses in detail and to establish whether there is a discontinuous dose response or threshold in activation of any important mediators of this response. In the two cell lines studied, we found a sensitive, linear dose response in early signaling and transduction pathways between doses of 0.1 and 2 Gy with no evidence of a threshold dose. We demonstrate that ATM-dependent signaling events to downstream targets including TP53, CHK1 and CHK2 occur after doses as low as 0.2 Gy and that these events promote an effective damage response. Using chemical inhibition of specific DNA repair enzymes, we show that inhibition of DNA-PK-dependent end joining has relatively little effect at low (<1 Gy) doses in hyper-radiosensitive cells and that at these doses the influence of RAD51-mediated repair events may increase, based on high levels of RAD51/BRCA2 repair foci. These data do not support a threshold model for activation of DNA repair in hyper-radiosensitive cells but do suggest that the balance of repair enzyme activity may change at low doses.     

The importance of appropriate controls, animal feed, and animal models in interpreting results from low-dose studies of bisphenol A

Interpreting results of studies that report only negative effects is problematic. A number of published studies to determine whether chemicals with estrogenic activity can cause effects at low doses have not taken into account the possibility that the commercial animal feed being used can mask effects of even potent estrogenic drugs such as diethylstilbestrol (DES). In addition, the sensitivity of the strain of animal being used for the specific category of chemical being tested has not always been described. For environmental chemicals, such as the estrogenic polycarbonate plastic monomer bisphenol A, DES is an appropriate positive control for estrogenic effects, and using an appropriate low dose of DES can eliminate the possibility of false-negative conclusions of safety when the above or other variables contribute to the negative outcome. Only when simultaneous positive effects of low doses of a positive control chemical such as DES and negative effects of environmentally relevant low doses of the test chemical are demonstrated within the same experiment are conclusions of no effect of the test chemical warranted, and this has not been reported for bisphenol A in any study. Instead, more than 90 refereed journal publications have reported effects due to exposure to low doses of bisphenol A in a wide variety of animals (for references see: http://rcp.missouri.edu/endocrinedisruptors/vomsaal/vomsaal.html). However, due to lack of attention to the importance of appropriate positive controls, a small number of studies reporting negative effects of bisphenol A have created a false sense of controversy regarding low-dose effects of bisphenol A.     

Benchmark dose calculation from epidemiological data

A threshold for dose-dependent toxicity is crucial for standards setting but may not be possible to specify from empirical studies. Crump (1984) instead proposed calculating the lower statistical confidence bound of the benchmark dose, which he defined as the dose that causes a small excess risk. This concept has several advantages and has been adopted by regulatory agencies for establishing safe exposure limits for toxic substances such as mercury. We have examined the validity of this method as applied to an epidemiological study of continuous response data associated with mercury exposure. For models that are linear in the parameters, we derived an approximative expression for the lower confidence bound of the benchmark dose. We find that the benchmark calculations are highly dependent on the choice of the dose-effect function and the definition of the benchmark dose. We therefore recommend that several sets of biologically relevant default settings be used to illustrate the effect on the benchmark results and to stimulate research that will guide an a priori choice of proper default settings.     

Investigation of the potentiation of the analgesic effects of fentanyl by ketamine in humans: a double-blinded, randomised, placebo controlled, crossover study of experimental pain[ISRCTN83088383]

BACKGROUND: Despite preclinical evidence suggesting a synergistic interaction between ketamine and opioids promoting analgesia, several clinical trials have not identified dosing regimens capable of eliciting a benefit in the co-administration of ketamine with opioids. METHODS: Ten healthy volunteers participated in a double blinded, randomised, placebo controlled, crossover laboratory study in order to determine whether a low dose of ketamine potentiated the antinociceptive effect of fentanyl without causing an increase in sedative effects. A battery of tests was used to assess both nociception and sedation including electrical current, pressure, thermal stimuli, psychometric tests, and both subjective and objective scores of sedation. Target controlled infusions of the study drugs were used. Ketamine and fentanyl were administered alone and in combination in a double-blinded randomised crossover design. Saline was used as the control, and propofol was used to validate the tests of sedation. Cardiovascular and respiratory parameters were also assessed. RESULTS: The electrical current pain threshold dose response curve of fentanyl combined with ketamine was markedly steeper than the dose response curve of fentanyl alone. While a ketamine serum concentration of 30 ng/ml did not result in a change in electrical pain threshold when administered alone, when it was added to fentanyl, the combination resulted in greater increase in pain threshold than that of fentanyl administered alone. When nociception was assessed using heat and pressure stimuli, ketamine did not potentiate the anti-nociceptive effect of fentanyl. There was no difference between the sedative effect of fentanyl and fentanyl in combination with ketamine as assessed by both subjective and objective measures of sedation. Cardiovascular and respiratory parameters were unaffected by the study drugs at the doses given. CONCLUSION: A serum concentration of ketamine that did not alter indices of sedation potentiated the antinociceptive effect of fentanyl. This potentiation of antinociception occurred without an increase in sedation suggesting that low steady doses of ketamine (30-120 ng/ml) might be combined with mu opioid agonists to improve their analgesic effect in a clinical setting. (296 words).     

New data on the effect of ionizing radiation on the growth of the aquatic plant <Emphasis Type="Italic">Elodea canadensis</Emphasis> in the laboratory

Toxicological experiments with radioactive bottom sediments and extrinsic γ-irradiation have demonstrated that the growth of common elodea roots is suppressed by irradiation at doses several times lower than the established threshold dose. The effect of γ-irradiation on the growth of elodea stems has not been observed at any dose used. The data obtained show that elodea could be recommended for use as an indicator of biological effects of radiation in the range of low radiation doses.     

Analysis of the epidemiological data concerning radiation carcinogenic effects and approaches to the low doses' upper limits determination in the aspect of a threshold of the unhealthy influences of ionizing radiation

The analysis of the epidemiological data regarding cancer mortality in cohorts of Japanese A-bomb survivors and Chermobyl liquidators exposed to different doses suggests that there are good reasons for recognizing the threshold of the radiocarcinogenic effect in the region of about 200 Gy (mSv). The analysis of solid cancer mortality in Japanese cohort, which exceeded the expected one in a dose diapason of 5-200 mSv, revealed a (quasi) plateau in a dose-effect curve and led to the conclusion that the nature of the overshoot is non-radiogenic. The analysis of supposedly dose dependent leucosis incidence in the limited low dose diapason in the Chernobyl cohort showed that the real coefficient of the excess absolute or relative radiation risk could not be received in the case because the larger part curve was placed under the control level. In supporting the principle of single hit in a cell nucleus as a base of microdosimetric determination of low radiation doses, the approach to objective delimitation between low, intermediate and high doses regions has been proposed. The low doses upper limit of sparse ionizing radiation for cell nucleus of 8 microns in diameter has been evaluated as 0.65 mGy. It can serve for evaluation of the dose rate threshold regarding the safe chronic radiation levels in the environment.     

Genotoxicity in the eyes of bystander cells

The controversial use of a linear, no threshold extrapolation model for low dose risk assessment has become even more so in light of the recent reports on the bystander phenomenon. The answer to the question as to which of the two phenomena, bystander versus adaptive response, is more important has practical implication in terms of low dose radiation risk assessment. In this review, genotoxicity is used as an endpoint to introduce the two phenomena, provide some insight into the mechanisms of bystander effect and to bridge the two low dose phenomena which operate in opposite directions: the bystander effect tends to exaggerate the effect at low doses, by communicating damage from hit to non-hit cells whereas the adaptive response confers resistance to a subsequent challenging dose by an initial low priming dose.     

Radiation-related cancer risks at low doses among atomic bomb survivors

To clarify the information in the Radiation Effects Research Foundation data regarding cancer risks of low radiation doses, we focus on survivors with doses less than 0.5 Sv. For reasons indicated, we also restrict attention mainly to survivors within 3, 000 m of the hypocenter of the bombs. Analysis is of solid cancer incidence from 1958-1994, involving 7,000 cancer cases among 50,000 survivors in that dose and distance range. The results provide useful risk estimates for doses as low as 0.05-0.1 Sv, which are not overestimated by linear risk estimates computed from the wider dose ranges 0-2 Sv or 0-4 Sv. There is a statistically significant risk in the range 0-0.1 Sv, and an upper confidence limit on any possible threshold is computed as 0.06 Sv. It is indicated that modification of the neutron dose estimates currently under consideration would not markedly change the conclusions.     

Low doses of biologically active substances: effects, possible mechanisms, and features

Some substances can produce a response in biological systems at extraordinarily low concentrations. Often called the 'low' or 'small' dose effect, the meaning or definition of what is meant by 'low' in this context is discussed. For the purposes of this article the expression is taken to be a concentration substantially below the equilibrium dissociation constant of the effector-target complex, but extremely low concentrations (<10(-19) m) are excluded from consideration. The main features of very low or 'small' dose action are described. Possible mechanisms of the effects of very low doses are suggested. The main thrust of the paper concerns the explanation of bimodal and polymodal dose-effect curves. A general formal kinetic model for such curves and its application will be discussed.     

Relationship between radiation induced adaptive response in human fibroblasts and changes in chromatin conformation

Chromatin conformation changes in the normal human fibroblasts VH-10 were studied by the method of anomalous viscosity time dependence (AVTD). Gamma-irradiation of cells in a dose range of 0.1–3 Gy caused an increase in maximal viscosity of cell lysates. Conversely, irradiation of cells with low doses of 0.5 or 2 cGy resulted in a decrease in the AVTD peaks with a maximum effect approximately 40 min after irradiation. The same exposure conditions were used to study a possible adaptive effect of low doses, measured by changes in cell survival. A primary dose of 2 cGy caused significant modification of cell response to a challenge dose. Approximately 20% protection to challenge doses of 0.5 Gy (p < 0.003), 2 Gy (p < 0.02) and 2.5 Gy (p < 0.002) was observed. However, the direction of this effect (adaptation or synergism) was found to be dependent on a challenge dose. The combined effect of 2 cGy and 1 Gy was significantly synergistic, while no modification was observed for 1.5 Gy and 3 Gy. A partial correlation was found between the AVTD changes and cell survival when the combined effect of a primary dose of 2 cGy and challenge dose was examined. The dose of 2 cGy alone increased survival by 16% (p < 0.0003). These results suggest that the low-dose induced effects on survival may be related to chromatin reorganization.     

Tumor induction and life shortening in BC3F1 female mice at low doses of fast neutrons and X rays

Extension of previous investigations at this laboratory regarding life shortening and tumor induction in the mouse has provided more complete dose-response information in the low dose region of X rays and neutrons. A complete observation of survival and late pathology has been carried out on over 2000 BC3F1 female mice irradiated with single doses of 1.5 MeV neutrons (0.5, 1, 2, 4, 8, 16 cGy) and, for comparison, of X rays (4, 8, 16, 32, 64, 128, 256 cGy). Data analysis has shown that a significant life shortening is observable only for individual neutron doses not lower than 8 cGy. Nevertheless, assuming a linear nonthreshold form for the overall dose-effect relationships of both radiation qualities, an RBE value of 12.3 is obtained for the 1.5 MeV neutrons. The induction of solid tumors by neutrons becomes statistically significant at individual doses from 8 cGy and by X rays for doses larger than 1 Gy. Linear dependence on neutron dose appears adequate to interpret the data at low doses. A separate analysis of ovarian tumor induction substantiates the hypothesis of a threshold dose for the X rays, while this is not strictly needed to interpret the neutron data. A trend analysis conducted on the neoplasm incidence confirms the above findings. Death rates have been analyzed, and a general agreement between the shift to earlier times of these curves and tumor induction was found.     

Defining EMS and ENU dose-response relationships using the Pig-a mutation assay in rats

In recent years, experimental evidence has accumulated that supports the existence of sublinear dose-response relationships at low doses of DNA reactive mutagens. However, creating the in vivo data necessary to allow for a more detailed dose-response modeling with the currently available tools might not always be practical. The purpose of the current work was to evaluate the utility of the Pig-a gene mutation assay to rapidly identify dose-response relationships for direct acting genotoxicants. The induction of mutations in the peripheral blood of rats was evaluated following 28 days of exposure down to low doses of the direct acting alkylating agents ethyl methane sulfonate (EMS) and ethylnitrosourea (ENU). Using statistical modeling based on the 28-day studies, a threshold for mutation induction for EMS was estimated to be 21.9mg/kg, whereas for the more potent ENU, the threshold was estimated to be 0.88mg/kg. Comparing mutation frequencies from acute and sub-chronic dosing indicated less than additive dose-response relationships, further confirming the possibility of a threshold dose-response relationship for both compounds. In conclusion, the work presented provides evidence that the Pig-a assay might be a practical alternative to other in vivo mutation assays when assessing dose-response relationships for direct acting mutagens and that an experimental approach using fractionated dosing could be used to substantiate a biological mechanism responsible for the observation of a sublinear dose-response relationship.     

Susceptibility differences in chemical carcinogenesis linearize the dose-response relationship: threshold doses can be defined only for individuals

The debate on thresholds in dose-response relationships for chemical carcinogenesis concentrates on the question of mechanisms of action that come into play only at dose levels that overwhelm compensatory control mechanism, such as DNA repair or regulation of cell proliferation and death. In this article, individual susceptibility differences are introduced. It is postulated that one single threshold dose cannot be defined for a heterogeneous population because both the background rate of carcinogenesis and specific exposure-related effects differ between individuals. A threshold dose can therefore be defined only on an individual basis and for a given organ. Expressed as a time-to-tumor, the threshold dose results in tumor manifestation at exactly the end of the specified observation period. For those individuals who do not have cancer by the end of this period, the dose was below their individual threshold dose, for those who do have cancer, the dose was above their threshold dose. Based on this concept, a distinction between genotoxic and non-genotoxic carcinogens is no longer required; both types modulate time-to-tumor. Although the present analysis does not allow to define a threshold dose for a population, the setting of a "limit value" for regulatory purposes can be considered if regulators are aware of the fact that this splits a population at some percentile into a group for which the chosen standard is protective and a group for which it might not be. Investigation of factors that confer particular susceptibility to individuals is the key to an understanding of the dose-response relationship at low dose.     

Low dose genotoxicity of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) in gpt delta transgenic mice

Although humans are chronically exposed to most environmental chemicals at low doses, genotoxicity assays with rodents are usually performed at high doses with short treatment period. To investigate the dose-response of genotoxicity at lower doses, gpt delta transgenic mice were fed a diet containing 300, 30 or 3 parts per million (ppm) of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) for 12 weeks and the gpt mutations in the liver were analyzed. In addition, the mice were continuously fed a diet containing MeIQx at a dose of 300 ppm for 78 weeks to examine the effect of a long-term treatment. In the mice treated for 12 weeks, the gpt mutant frequencies (MFs) were 8.6-, 2.3- and 1.2-fold higher than the control level at the doses of 300, 30 and 3 ppm, respectively. G:C to T:A transversion was the most predominant type of mutations and the fold increases in the specific MF of G:C to T:A were 58.2, 4.4 and 1.7 above the control at the three doses, respectively. The increases in the whole gpt and specific MFs at 3 ppm were not statistically significant. In the mice treated with 300 ppm of MeIQx for 78 weeks, the gpt MF was about 20 times higher than that of the untreated mice fed a control diet for 78 weeks, which was about two times higher than that of the untreated mice at 12 weeks. These results suggest that no obvious genotoxic effects can be detectable at the dose of MeIQx at 3 ppm in the liver and a longer treatment substantially enhances the genotoxicity. Factors constituting the practical threshold dose are discussed.     

Neoplastic transformation in vitro by low doses of ionizing radiation: role of adaptive response and bystander effects

The shape of the dose-response curve for cancer induction by low doses of ionizing radiation is of critical importance to the assessment of cancer risk at such doses. Epidemiologic analyses are limited by sensitivity to doses typically greater than 50-100 mGy for low LET radiation. Laboratory studies allow for the examination of lower doses using cancer-relevant endpoints. One such endpoint is neoplastic transformation in vitro. It is known that this endpoint is responsive to both adaptive response and bystander effects. The relative balance of these processes is likely to play an important role in determining the shape of the dose-response curve at low doses. A factor that may influence this balance is cell density at time of irradiation. The findings reported in this paper indicate that the transformation suppressive effect of low doses previously seen following irradiation of sub-confluent cultures, and attributed to an adaptive response, is reduced for irradiated confluent cultures. However, even under these conditions designed to optimize the role of bystander effects the data do not fit a linear no-threshold model and are still consistent with the notion of a threshold dose for neoplastic transformation in vitro by low LET radiation.     

The effect of dose rate on radiation-induced neoplastic transformation in vitro by low doses of low-LET radiation

The dependence of the incidence of radiation-induced cancer on the dose rate of the radiation exposure is a question of considerable importance to the estimation of risk of cancer induction by low-dose-rate radiation. Currently a dose and dose-rate effectiveness factor (DDREF) is used to convert high-dose-rate risk estimates to low dose rates. In this study, the end point of neoplastic transformation in vitro has been used to explore this question. It has been shown previously that for low doses of low-LET radiation delivered at high dose rates, there is a suppression of neoplastic transformation frequency at doses less than around 100 mGy. In the present study, dose-response curves up to a total dose of 1000 mGy have been generated for photons from (125)I decay (approximately 30 keV) delivered at doses rates of 0.19, 0.47, 0.91 and 1.9 mGy/min. The results indicate that at dose rates of 1.9 and 0.91 mGy/min the slope of the induction curve is about 1.5 times less than that measured at high dose rate in previous studies with a similar quality of radiation (28 kVp mammographic energy X rays). In the dose region of 0 to 100 mGy, the data were equally well fitted by a threshold or linear no-threshold model. At dose rates of 0.19 and 0.47 mGy/min there was no induction of transformation even at doses up to 1000 mGy, and there was evidence for a possible suppressive effect. These results show that for this in vitro end point the DDREF is very dependent on dose rate and at very low doses and dose rates approaches infinity. The relative risks for the in vitro data compare well with those from epidemiological studies of breast cancer induction by low- and high-dose-rate radiation.     

The effects of neutrons in Hiroshima. Implications for the risk estimates

Risk estimates for radiation-induced late effects are relevant to various considerations in radiation protection. Most of these considerations relate to small doses for which no excess risk can be seen even in extensive epidemiological studies. Risk coefficients for radiation protection must, therefore, be based on uncertain extrapolation of observations obtained at moderate or high doses. The extrapolation can not be replaced, as yet, by new, more direct information on processes such as radiation-induced genetic instability or adaptive response. While the new findings indicate complexities that may be highly relevant to the effectiveness- or lack of effectiveness- of radiation at low doses, they remain insufficiently understood to permit a decision as to whether dose-effect relations are linear, curvilinear, or have a threshold in dose. In view of these uncertainties radiation-protection regulations are, today, based on the conservative assumption of a linear dose dependence without threshold. This approach assures a sufficient degree of protection, but it may become unreasonably over-conservative, when the cautious hypothesis is treated as proven fact, and when-in addition-the assumed initial slope of the dose relation is not critically evaluated. A reliable evaluation needs to be based on the follow-up of the atom-bomb bomb survivors, and several major aspects of current interest are discussed here. a) Mortality from solid tumours in Hiroshima shows a statistically significant excess at a colon dose of 50 mGy; however, it is likely that this is the result of a bias in assigning causes of death. b) The solid cancer mortality data of the atom-bomb survivors are consistent with linearity in dose, but they can be shown to be equally consistent with a considerable degree of curvature. c) Even with the present dosimetry system, DS86, a substantial part of the effect at small doses in Hiroshima could be due to neutrons. If this is the case, the risk estimates for gamma-rays need to be accordingly decreased. d) Numerous neutron-activation measurements in Hiroshima indicate that the DS86 underestimates the neutron doses. The evidence is, up to now, based only on activation products of low energy neutrons, but efforts are currently underway to determine activation products of high energy neutrons. If these measurements should substantiate the present trend, the cancer data in Hiroshima would cease to be reliable proof of an effect of gamma-rays at low doses. Instead the dose dependence for gamma-rays could be purely quadratic, and any initial slope in the linear-quadratic dependence might well be attributable to neutrons only.     

Glycophorin A mutant frequency in radiation workers at the nuclear power plants and a hospital

We studied to assess the validity of the glycophorin A (GPA) mutant assay as a biological marker of the cumulative effects of chronic low doses of ionizing radiation. In 144 nuclear power plants workers and 32 hospital workers, information on confounding factors, such as age and cigarette smoking, was obtained through a self-administered questionnaire. The information on physical exposure doses was obtained from the registries for radiation exposure monitoring and control at each facility. The range of cumulative exposure doses were 0-12.02cGy. GPA mutant assay was performed by the BR6 method with modification using a FACScan flow cytometer. Potential confounders, such as, age and cigarette smoking habits showed increasing trends with GPA variants, but were not of statistical significance. The hospital workers showed higher frequency of the GPA NO variant than nuclear power plant workers. Significant dose-response relationships were found between cumulative exposure to radiation and variants levels by simple and multiple linear regression models. The slope of regression equation of the dose-response of nuclear power plants workers was much smaller than that of hospital workers. These findings suggest that there may be dose-rate effects. In a population exposed to chronic low-dose radiation, the GPA assay shows potential to be used as an effective biologic marker for assessing the cumulative exposure dose although it could not be able to see a dose relation below 10cGy of cumulative exposure dose.     

Prostaglandin release by the chick embryo heart is increased by 2,3,7,8-tetrachlorodibenzo-p-dioxin and by other cytochrome P-448 inducers

Exposure of chick embryos in ovo to cytochrome P-448 inducers 2,3,7,8-tetrachlorodibenzo-p-dioxin, 3,4,3',4'-tetrachlorobiphenyl, 3,4,5,3',4',5'-hexachlorobiphenyl and beta-napthoflavone, increased cardiac prostaglandins in vitro. The dose response relationships were biphasic with prostaglandin release increasing at the low doses and returning to basal levels at higher doses. Phenobarbital was ineffective. Increased cardiac prostaglandin release was detected at doses that induced hepatic 7-ethoxyresorufin deethylase (7-ER) but which were below the threshold for cardiac induction. The fall in prostaglandin release coincided with induction of cardiac 7-ER and therefore may be attributable to increased prostaglandin metabolism. These studies show that the P-450 system may interact with the arachidonic acid metabolizing system to increase PG release and that this effect may be part of the pleiotypic response to Ah-receptor activation.