bluuebunny/crossref_metadata_2025 huggingface dataset:
Simultaneous quantitative determination of cyclosporine A and its three main metabolites (AM1, AM4N and AM9) in human blood by liquid chromatography/mass spectrometry using a rapid sample processing method
Authors: Koseki Nozomu , Nakashima Akinori , Nagae Yusuke , Masuda Naoki
Published: February 2006

We have developed a sensitive and specific liquid chromatography/mass spectrometry (LC/MS) method for the simultaneous determination of cyclosporine A (CsA) and its three main metabolites (AM1, AM4N and AM9) in human blood. Following protein precipitation, supernatant was directly injected into the LC/MS system. Chromatographic separation was accomplished on a Symmetry C8 (4.6 × 75 mm, 3.5 µm) column with a linear gradient elution prior to detection by atmospheric pressure chemical ionization (APCI) MS using selected ion monitoring (SIM) in positive mode. This method can be applied to single mass equipment. The analytical range for each analyte was set at 1–2500 ng/mL using 100 µL of blood sample. The analytical method was fully validated according to FDA guidance. Intra‐day mean accuracy and precision were 95.2–113.5% and 0.9–8.9%, respectively. Inter‐day mean accuracy and precision were 95.8–107.0% and 1.5–10.7%, respectively. In blood all analytes were stable during three freeze/thaw cycles, for 24 h at room temperature and for 12 months at or below −15°C. Stability was also confirmed in processed samples for 24 h at 10°C and for 6 months at 4°C in methanol. In addition, we confirmed the method could avoid matrix effects from transplant subjects' samples. This LC/MS technique provided an excellent method for simultaneous quantitative determination of CsA and its three metabolites for evaluation of their pharmacokinetic profiles. Copyright © 2006 John Wiley & Sons, Ltd.
2,5‐Dihydroxyacetophenone: a matrix for highly sensitive matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometric analysis of proteins using manual and automated preparation techniques
Authors: Wenzel Thomas , Sparbier Katrin , Mieruch Thorsten , Kostrzewa Markus
Published: February 2006

2,5‐Dihydroxyacetophenone (DHAP) is presented as a matrix which enables highly sensitive matrix‐assisted laser desorption/ionization time‐of‐flight (MALDI‐TOF) mass spectrometric analysis of peptides, proteins and glycoproteins on AnchorChip targets. Depending on the protein, lower fmol amounts can be detected due to the increased homogeneity and concentration of the crystallization of the analyte/matrix mixture on the anchors. Best results could be generated in the mass range of 8–100 kDa. All sample/matrix preparation steps starting from mixing of DHAP matrix solution with sample solution to the transfer of the mixture to the MALDI‐TOF target can be performed manually or automatically allowing low‐ and high‐throughput analyses. Copyright © 2006 John Wiley & Sons, Ltd.
Skills training in telerobotic surgery
Authors: Hance J , Aggarwal R , Undre S , Darzi A
Published: February 2006

Surgical robots have the potential to expand the repertoire of minimally invasive surgery resulting in more patients benefiting from lower operative morbidity and shorter hospital stays. However, in a similar manner to all new surgical interventions it necessary to explore the learning curves of practitioners as they adopt this new technology to enable optimisation of future training programs. Only when the standard of practice is firmly established, should the proliferation of robotic practitioners be encouraged thus ensuring patient safety is not compromised. Copyright © 2005 Robotic Publications, Ltd.
Robotics in urology: an update
Authors: Nedas T G , Challacombe B J , Dasgupta P
Published: February 2006

Urology has been quick to realise the advantages of surgical robots. We describe the main devices currently in use, the advantages and disadvantages of robotic‐assisted surgery and the current and potential urological applications focussing on robot‐assisted radical prostatectomy. Copyright © 2005 Robotic Publications Ltd.
Respiration tracking in radiosurgery without fiducials
Authors: Schweikard A , Shiomi H , Adler J
Published: February 2006

In robotic radiosurgery, a focused beam of radiation is moved by a robot arm. We investigated methods for soft‐tissue navigation using robotic radiosurgery. In previous work we described a method for real‐time tracking based on correlation between the motion of implanted fiducial markers and external skin markers. In this work we extend our method of correlation‐based tracking to tracking without implanted fiducials. We propose to use deformation algorithms on CT data sets combined with registration of digitally reconstructed radiographs and intra‐treatment X‐ray images to obtain intermittent information on the target location. This information is then combined with our basic correlation method to achieve real‐time tracking. Our study investigates the feasibility of this approach from the point of view of computing time and required level of user interaction. The term 7D registration is coined to describe the underlying method for performing this task. Copyright © 2005 Robotic Publications, Ltd.
Disease cluster detection: a critique and a Bayesian proposal
Authors: Lawson Andrew B.
Published: February 2006

This paper reviews issues in the analysis of non‐focussed clustering, and proposes a novel approach to cluster modelling that can be used in a surveillance context. The novel approach involves the use of local likelihood models for the analysis of clustering in small area health data. Local likelihood is used when interdependence between data events at locations is modelled directly, as opposed to the modelling of a hidden process of cluster centres. This approach allows the use of conventional posterior sampling. It also allows a less parameterized approach to the form of clusters detected. The idea of a spatially dependent lasso which provides the local maxima for the aggregation of locations is considered as an approximation. The methods are applied to a well known data set and compared with Satscan, and a conditional logistic Bayesian model. Copyright © 2006 John Wiley & Sons, Ltd.
Improved hypothesis testing for coefficients in generalized estimating equations with small samples of clusters
Authors: McCaffrey Daniel F. , Bell Robert M.
Published: February 2006

The sandwich standard error estimator is commonly used for making inferences about parameter estimates found as solutions to generalized estimating equations (GEE) for clustered data. The sandwich tends to underestimate the variability in the parameter estimates when the number of clusters is small, and reference distributions commonly used for hypothesis testing poorly approximate the distribution of Wald test statistics. Consequently, tests have greater than nominal type I error rates. We propose tests that use bias‐reduced linearization, BRL, to adjust the sandwich estimator and Satterthwaite or saddlepoint approximations for the reference distribution of resulting Wald t‐tests. We conducted a large simulation study of tests using a variety of estimators (traditional sandwich, BRL, Mancl and DeRouen's BC estimator, and a modification of an estimator proposed by Kott) and approximations to reference distributions under diverse settings that varied the distribution of the explanatory variables, the values of coefficients, and the degree of intra‐cluster correlation (ICC). Our new method generally worked well, providing accurate estimates of the variability of fitted coefficients and tests with near‐nominal type I error rates when the ICC is small. Our method works less well when the ICC is large, but it continues to out‐perform the traditional sandwich and other alternatives. Copyright © 2006 John Wiley & Sons, Ltd.
A maximum likelihood method for studying gene–environment interactions under conditional independence of genotype and exposure
Authors: Cheng K. F.
Published: February 2006

Given the biomedical interest in gene–environment interactions along with the difficulties inherent in gathering genetic data from controls, epidemiologists need methodologies that can increase precision of estimating interactions while minimizing the genotyping of controls. To achieve this purpose, many epidemiologists suggested that one can use case‐only design. In this paper, we present a maximum likelihood method for making inference about gene–environment interactions using case‐only data. The probability of disease development is described by a logistic risk model. Thus the interactions are model parameters measuring the departure of joint effects of exposure and genotype from multiplicative odds ratios. We extend the typical inference method derived under the assumption of independence between genotype and exposure to that under a more general assumption of conditional independence. Our maximum likelihood method can be applied to analyse both categorical and continuous environmental factors, and generalized to make inference about gene–gene–environment interactions. Moreover, the application of this method can be reduced to simply fitting a multinomial logistic model when we have case‐only data. As a consequence, the maximum likelihood estimates of interactions and likelihood ratio tests for hypotheses concerning interactions can be easily computed. The methodology is illustrated through an example based on a study about the joint effects of XRCC1 polymorphisms and smoking on bladder cancer. We also give two simulation studies to show that the proposed method is reliable in finite sample situation. Copyright © 2006 John Wiley & Sons, Ltd.
Nested frailty models using maximum penalized likelihood estimation
Authors: Rondeau V. , Filleul L. , Joly P.
Published: February 2006

The frailty model is a random effect survival model, which allows for unobserved heterogeneity or for statistical dependence between observed survival data. The nested frailty model accounts for the hierarchical clustering of the data by including two nested random effects. Nested frailty models are particularly appropriate when data are clustered at several hierarchical levels naturally or by design. In such cases it is important to estimate the parameters of interest as accurately as possible by taking into account the hierarchical structure of the data. We present a maximum penalized likelihood estimation (MPnLE) to estimate non‐parametrically a continuous hazard function in a nested gamma‐frailty model with right‐censored and left‐truncated data. The estimators for the regression coefficients and the variance components of the random effects are obtained simultaneously. The simulation study demonstrates that this semi‐parametric approach yields satisfactory results in this complex setting. In order to illustrate the MPnLE method and the nested frailty model, we present two applications. One is for modelling the effect of particulate air pollution on mortality in different areas with two levels of geographical regrouping. The other application is based on recurrent infection times of patients from different hospitals. We illustrate that using a shared frailty model instead of a nested frailty model with two levels of regrouping leads to inaccurate estimates, with an overestimation of the variance of the random effects. We show that even when the frailty effects are fairly small in magnitude, they are important since they alter the results in a systematic pattern. Copyright © 2006 John Wiley & Sons, Ltd.
Optimum blood sampling time windows for parameter estimation in population pharmacokinetic experiments
Authors: Graham Gordon , Aarons Leon
Published: February 2006

Clinical trials requiring the collection of pharmacokinetic information often specify blood samples to be taken at fixed times. This may be feasible when trial participants are in a controlled environment such as in early phase clinical trials, however it becomes problematic in trials where patients are in an out‐patient clinic setting such as in late phase drug development. In such a situation it is common to take blood samples when it is convenient for all involved and may result in data that are uninformative. This paper proposes an approach to pharmacokinetic study design that allows greater flexibility as to when blood samples can be taken and still result in data that allows satisfactory parameter estimation. The sampling window approach proposed in this paper is based on determining time intervals around theD‐optimum pharmacokinetic sampling times. These intervals are determined by allowing the sampling window design to result in a specified level of efficiency when compared to the fixed timesD‐optimum design. Several approaches are suggested for dealing with this design problem. Copyright © 2006 John Wiley & Sons, Ltd.
NMR Diffusometry and Dynamic Light Scattering Studies of Amylopectin: Effect of Shearing and Heating on the Size Distribution and Diffusion Behaviour
Authors: Wassenius Helena , Löfroth Jan‐Erik , Nydén Magnus
Published: February 2006

The influence of different factors on the diffusion behaviour of cationized potato amylopectin (CAP) in water was investigated by NMR Diffusometry and Dynamic Light Scattering (DLS). The sample preparation procedure was found to have a fundamental influence on the size and size distribution evolution of the different samples, whereas the addition of NaN3 or the replacement of CAP by non‐cationic potato amylopectin (PAP) did not influence the diffusion behaviour. NMR samples were prepared by re‐dissolving a freeze‐dried CAP sample in D2O at ambient temperature or by heating. Some samples were sheared, reducing their average aggregate size by a factor of 40. The unheated samples had a large average molecular size, indicating incomplete aggregate dissolution. The single‐fit average diffusion coefficient Dav,init showed a strong time evolution for the unheated samples, increasing with a factor of 1000 over 20 days for both sheared and unsheared samples. This effect was mainly attributed to incomplete sample dissolution, leading to a time evolution of the molecular size and size distribution. For heated samples, Dav,init oscillated around a much higher value indicating the occurrence of sub‐molecular sized fragments and possibly also a time‐coherent rearrangement of the sample. The size distribution was very large for both heated and unheated samples, with σlnD values of up to 2.5 and diffusion coefficients ranging from 10‐14 to 10‐11 m2/s in the same sample. DLS measurements corroborated the conclusions drawn from the results found by NMR diffusometry, but the complementary use of DLS and NMR diffusometry also highlighted the difference between the two methods for size determination. With NMR diffusometry, the proportion of small molecules may be slightly exaggerated, whereas the scattering methods mostly used in previous studies may underestimate their importance, resulting in a larger mean aggregate size than may actually be the case. This study thus draws the attention to the importance of the analysis method for the size and size distribution of amylopectin samples.
The Influence of Thiocyanate Ions on the Formation of the Starch‐Iodine Complex
Authors: Cornell Hugh J. , Rix Colin J.
Published: February 2006

The redox reaction between thiocyanate and iodine species present in an aqueous solution of KI results in the formation of thiocyanogen, (SCN)2. This reaction interferes with the formation of inclusion complexes of a potato amylose with iodine species such as I3‐ and I5‐. As these ions react with thiocyanate ions (SCN‐) the blue colour of the amylose‐iodine complex diminishes. The rate of this decrease depends upon the concentration of both the SCN‐ and the iodine, and appears to follow pseudo‐second order kinetics. Using a UV/visible spectrophotometer the reaction of iodine with KSCN can be followed, with or without amylose, and shows a lowering in concentration of iodine species absorbing at 290–300 nm, together with a lowering in absorbance at 630 nm, due to a lesser amount of the amylose‐iodine complex. Interference with iodine complex formation was also noted with a potato amylopectin. The technique provides the basis for the study of other reactions of iodine such as that with selenocyanate (SeCN‐).
New approaches to mutagenicity studies in animals for carcinogenic and mutagenic agents. I. Modification of the heritable translocation test
Authors: Adler Ilse‐Dore
Published: February 2006

Two alternative modifications of the experimental protocol for the heritable translocation test are described. One of them proposes to mate F1 males and F1 females within one experimental group and eliminate normal pairs by a sequential decision procedure based on litter sizes. Pairs that do not meet the criteria for normal litter size have to be separated and tested against normal partners. Male translocation suspects are analyzed cytogenetically for the presence of a reciprocal translocation. Female translocation suspects or XO suspects are verified through analysis of their male and female progeny. The second modification of the heritable translocation test omits fertility testing and proposes to cytogenetically analyze 25 diakineses‐metaphases I from each F1 male in the test.

Using either one or both protocols it was shown that 20 mg/kg of methyl methanesulfonate (MMS) induced 1.3% heritable translocations in late spermatids and early spermatozoa. With 2.5 mg/kg of mitomycin C, 0.3% and 0.4% translocation carriers were recovered from treated primary spermatocytes and early spermatids, respectively. A dose of 300 R gamma rays resulted in 1.6% translocations in spermatozoa and 4.8% translocations in primary spermatocytes.

The advantage of the modified fertility test lies in the doubling of the sample size by including the females. However, a disadvantage is the amount of time and labor that is necessary to verify female translocation carriers. The cytogenetic translocation test protocol with all F1 males is considered more reliable and faster. It lacks the possibility of error entailed in the decision procedure by fertility testing, and it requires less time, personnel, and animal space.
Dominant lethality in Xenopus laevis induced with triethylenemelamine (TEM)
Authors: McKinnell Robert G. , Kren Betsy T. , Bergad Robert , Schultheis Melissa , Byrne Timothy , Schaad J. W.
Published: February 2006

Adult male South African clawed toads, Xenopus laevis, were injected intraperitoneally with triethylenemelamine (TEM) dissolved in water over a dose range of 13–1,300 μg/kg or with water (controls). Mating of the treated males with untreated females was induced seven days later by injection of human chorionic gonadotropic hormone (HCG). Accumulated lethality one week after fertilization among progeny was dose related. Morphological abnormalities among embryos that survived to hatching were similarly dose related. Because anatomically anomalous embryos may have impaired behavior, swimming capacity was tested, and anomalies in swimming behavior were also found to be dose related. Finally, short‐term cell cultures were made on minced embryos to obtain chromosome spreads. Structural aberrations, rings, dicentrics, etc of somatic chromosomes were also dose related.
Tumor promoter 12‐0‐tetradecanoylphorbol 13‐acetate mimics epidermal growth factor‐induced precocious tooth eruption in the rodent
Authors: Sim F. Russell P. , Salomon David S. , Nylen Marie U. , Pratt Robert M.
Published: February 2006

Daily injections of epidermal growth factor (EGF) or the tumor promoter, 12‐0‐tetradecanoylphorbol 13‐acetate (TPA) to neonatal Swiss Webster S/W (NIH) mice caused precocious tooth eruption. However, TPA, unlike EGF, did not enhance eyelid opening. The response was correlated with the tumor‐promoting activity of various phorbol congeners. Furthermore, TPA‐induced tooth eruption appeared to be prostaglandin‐dependent, whereas EGF‐induced eruption was insensitive to the prostaglandin inhibitors.
Genetic effects of procarbazine in the yeast Saccharomyces cerevisiae, strain D4
Authors: Frezza Domenico , Bianchi Vera
Published: February 2006

Procarbazine (PCZ) was tested for its ability to induce mitotic gene conversions at the ade and trp loci of Saccharomyces cerevisiae, strain D4. The influence of the following factors was examined: growth phase of the yeast cells (log vs stationary phase), pH of the treatment solution, and addition of mouse S9 fractions prepared from different organs. The drug was found more toxic and mutagenic at low doses (up to 25 mg/ml) for log phase cells, and scarcely toxic but highly mutagenic, even at high doses, for stationary phase cells. PCZ activity was reduced by acidic pH, and suppressed by S9 mix.

Gene conversions were also analyzed in the intrasanguineous host‐mediated assay performed in mice orally administered with PCZ. In such conditions PCZ was ineffective in stimulating mitotic gene conversions, probably owing to its inactivation in the acidic environment of the gastroenteric tract.
Genetic effects of drug interaction in tuberculosis patients and their fate
Authors: Jaju Madhuri , Jaju Manjula , Ahuja Y. R.
Published: February 2006

In this paper we will discuss the genetic consequences of drug interaction in tuberculosis patients. Blood from tuberculosis patients was cultured before, during, and after withdrawal of therapy involving five different drug combinations of isoniazid (INH), thiacetazone (TAZ), para‐aminosalicylic acid (PAS), and streptomycin (SM). The approaches used to detect DNA damage were chromosome aberrations and sister chromatid exchanges (SCEs). A total of 179 subjects were analyzed. In combination these drugs showed synergistic, additive, and antagonistic effects, though they were found to be nonclastogenic individually. Four of the drug combinations, INH+TAZ, INH+PAS, INH+TAZ+SM, and INH + PAS + SM, induced a significant increase in the frequency of aberrations, whereas INH+SM did not induce aberrations. In fact, SM appeared to reduce the frequency of aberrations. SCEs were increased in only two patients: one treated with INH + TAZ and the other with INH + PAS. The frequency of aberrations after withdrawal of therapy was decreased; it was slightly higher than the controls, though it was insignificant. The return to normalcy could be due to elimination of damaged cells or the repair of DNA in lymphocytes. Though the drug‐induced aberrations do not persist after withdrawal of therapy, the chromosome damaging combinations of drugs should be used with caution, because the possibility of meiotic chromosome damage in germ cells (during therapy), which might be passed on to the next generation, cannot be ruled out.
Lack of in vivo and in vitro genotoxicity with the nonsteroid, antiinflammatory agent sodium meclofenamate
Authors: Kropko Melvin L. , Fitzgerald J. E. , De la Iglesia Felix A.
Published: February 2006

Sodium meclofenamate (Meclomen®), CI‐583, is an anthranilic acid salt developed as a nonsteroidal, antiinflammatory agent. A multitest battery of short‐term tests was employed to characterize the genotoxic and mutagenic potential of the compound by measuring point mutations in bacteria, induction of sister‐chromatid exchange, chromosome aberrations, and gene mutations in mammalian cells in vitro. In vitro assays included metabolic activation. The in vivo assay was for chromosome aberrations in bone marrow cells from rats. At toxicity‐limited doses for each assay, no activity was detected in the bacterial or mammalian cell mutation assays, and sister‐chromatid exchange frequencies were not increased over control rates. When sodium meclofenamate was evaluated in vitro, chromosome aberrations were induced under metabolic activation in CHO cells. Chromosome aberrations and clastogenic activity were not demonstrated after oral administration of Meclomen® to male rats. It was concluded that sodium meclofenamate does not possess overt mutagenic potential under these conditions. The activity seen in vitro with CHO cells after metabolic activation did not correlate with the results from the other tests and was attributed to the formation of reactive metabolites not present or formed in in vivo systems.
An extended evaluation of an in vivo teratology screen utilizing postnatal growth and viability in the mouse
Authors: Gray Leon Earl , Kavlock Robert J.
Published: February 2006

A teratology test system proposed by Chernoff and Kavlock [3,4] utilizes growth and viability for 3 days after birth to prioritize chemicals for standard teratology testing. The present study is an extended observation of the growth and viability of Chernoff and Kavlock's animals from 41 treatments for 250 days to determine if neonatal weight reductions persisted throughout life, if mortality occurred later in life or, if other serious abnormalities developed that could not be anticipated from the neonatal data. One chemical which was positive in the present study would be a false negative in the standard teratology test and the Chernoff/Kavlock Assay (CKA). Congo red did not cause frank malformations or mortality but it specifically altered reproductive function in the offspring. Two chemicals produced a variety of unusual abnormalities that were not apparent in the neonates, indicating that postnatal testing is necessary to determine the full teratogenic potential of some compounds. Mice exposed prenatally to cytosine arabinoside became hydrocephalic and 30% of them lacked lower incisors. Nitrofen caused hydrocephaly, the age of eye‐opening was delayed, a few of the mice had no eyes, and most of the mice lacked Harderian glands. Eighteen of the 41 treatments caused mortality of 15%, or greater, by day 3, and four of these treatments resulted in additional mortality past the neonatal period. No compound caused a decrease in viability after the neonatal period without also reducing days 1 or 3 litter sizes, validating the use of this measurement in the CKA. Cytosine arabinoside was administered later in gestation than the CKA dosing regime and caused a marginally significant increase in mortality near birth but killed over 50% of the pups after weaning. Altering the duration or period of exposure in the CKA may necessitate a longer postnatal evaluation than presently recommended [3,4]. Neonatal weight, the second parameter used in the CKA to prioritize chemicals for additional testing, appears to be less useful than viability. It was a poor predictor of subsequent growth, some early weight effects persisted, while most of those concurrent with maternal weight reductions were transient. A correlation analysis of the parameters measured in the CKA and in the present study, excluding the compounds that produce frank malformations, indicated that compounds that reduced maternal weight gain during dosing had fewer live pups and growth was transiently retarded. Compounds could be tested in the CKA at a dose that result in less than a 20% reduction in maternal weight gain during dosing without confounding maternal toxicity with postnatal effects.
Homogeneous populations of Artemia nauplii and their potential use for in vitro testing in developmental toxicology
Authors: Sleet R. B. , Brendel K.
Published: February 2006

Efforts have begun to establish test subjects other than the intact pregnant mammal to serve as models for rapid teratology screens. Artemia nauplii transcending instar I to later instars were examined to determine their potential for indicating chemicals as potential developmental hazards and thus prioritizing them for more extensive in vivo testing. Several criteria selected for assessing the system's potential for screening were the ability to: 1) collect homogeneous populations of instar I nauplii; 2) characterize intermediate development by technically simple measurements; and 3) demonstrate development‐related differentials in naupliar vulnerability.

Homogeneous populations of nauplii were harvested from a flow‐through hatching and cold storage system. Nauplii accumulated in the system are stored at 4°C in a quiescent state with little physical (body length, body water volume) and biochemical (DNA and protein levels) change and thus are maintained at instar I. Intermediate development of nauplii transcending instar I to IV was characterized after the onset of 25°C incubation by measuring changes in drinking activity, body length, body water volume, and DNA and protein levels. During the first day of incubation, development was greatest between 6 and 24 hours of incubation.

Development‐related differentials in naupliar vulnerability were shown by comparing median lethal concentrations (LC50) estimated for cadmium sulfate (CdSO4), mercuric chloride (HgCl2), and sodium azide (NaN3) at various times during incubation. With cadmium and mercury, LC50s decreased as nauplii aged and developed; whereas, with azide, LC50s did not vary. Developing nauplii were differentially vulnerable to cadmium and inorganic mercury. Artemia nauplii transcending instar I to IV appear useful for indicating chemicals that preferentially interact with developmental events.
Teratogenic effects of carcinogenic agents on limb regeneration in the japanese newt Cynops pyrrhogaster
Authors: Pfeiffer Carl J. , Nagai Toshihiro , Fujimura Masaki , Tobe Takayoshi
Published: February 2006

Normal regeneration of the amputated forelimb of the Japanese newt Cynops pyrrhogaster and regeneration after a single intraperitoneal injection of three potent mutagenic/carcinogenic agents was investigated. Three dose levels of each agent and controls were tested for teratogenicity in this newt model with the following chemicals: N‐methyl‐N′‐nitro‐N‐nitrosoguanidine (MNNG), 4‐nitro‐quinoline‐1‐oxide, and 2‐(2‐furyl)‐3‐(5‐nitrofuryl)acrylamide. These chemicals were administered at 10 days (late dedifferentiation stage), 20 days (late bud stage), and 30 days (early digits stage) after amputation at the midforelimb. A total of 628 newts, with 16–20 animals per group, were used. Normal forelimb regeneration in Cynops pyrrhogaster closely paralleled that reported for other species. A variety of deformities, including syndactyly, polydactyly, oligodactyly, brachydactyly, and digital branching, were occasionally observed in control regenerating forelimbs, with syndactyly occurring at highest incidence (17.5%).

All three mutagens at all tested dose levels enhanced the incidence of teratogenic changes, though increases were not always statistically significant. MNNG, particularly when administered at the time of initial chondrogenesis (20 days, late bud stage), was especially teratogenic. The type of forelimb deformity was not mutagen‐specific in this experiment. As Cynops pyrrhogaster is easily and inexpensively maintained and tolerates surgery well, this model with the regenerating forelimb should prove useful for further studies on teratogen screening. Also, studies directed toward mutagenic and epigenetic effects of exogenous agents on rapidly proliferating and differentiating tissues can be investigated with this model, which obviates transplacental excursion and metabolism of test compounds.
Suppression by cyclohexanetriones of retinoic acid‐induced cartilage degradation in vitro and teratogenicity in vivo
Authors: Kistler Andreas
Published: February 2006

In cultured fetal rat bones, cyclohexanetriones that stimulate prostaglandin synthesis inhibited retinoic acid‐induced cartilage degradation in a dose‐dependent manner. The inhibition by the cyclohexanetrione Ro 31‐0521 was reversible, indicating that the effect was not due to cytotoxicity. Excess retinoic acid is teratogenic in rats and adversely affects the normal differentiation of various morphogenetic systems, depending on the time of administration. The following retinoic acid‐induced malformations were suppressed by Ro 31‐0521: malformations of long bones and of apical phalanges induced on days 13 and 15 of gestation, respectively; spina bifida and tail malformations induced on day 11 of gestation and cleft palate induced on day 15 of gestation. However, cleft palate and other head malformations including exencephaly induced by retinoic acid on day 11 of gestation were not suppressed but even increased by Ro 31‐0521. At a high dose, Ro 31‐0521 given alone on day 11 of gestation was embryolethal and teratogenic but was not on the tested other days, indicating that the cyclohexanetrione at specific stages and doses also interfered with normal morphogenesis like retinoic acid. Assuming that stimulation of prostaglandin synthesis is the main biological effect of the cyclohexanetriones, our findings suggest that prostaglandins may be involved in mediating retinoid action.
Co‐culture of rat embryos and hepatocytes: In vitro detection of a proteratogen
Authors: Oglesby Linda A. , Ebron Marian T. , Beyer Patricia E. , Carver Brenda D. , Kavlock Robert J.
Published: February 2006

The technique of whole embryo culture developed by New [Environ Health Perspect 18:105–110, 1976] provides a sensitive assay to evaluate the effects of a test chemical on embryo development independent of maternal influences. To detect proteratogens, this assay must be coupled with an exogenous metabolic activation system. We have developed methods for the co‐cultivation of rat embryos with primary hepatocytes, which offers several advantages over subcellular fractions when providing metabolic activation for in vitro assays. In the present study, rat embryos removed from the dam on day 10 of pregnancy were cocultivated in vitro with primary cultures of rat, rabbit, or hamster hepatocytes. Embryos co‐cultivated with hepatocytes developed normally, as did embryos exposed to a test chemical, cyclophosphamide (CP) in the absence of hepatocytes. When embryos were co‐cultivated with hepatocytes and exposed to CP, a doserelated embryotoxicity was observed, indicating metabolic activation of the proteratogen. Using hepatocytes isolated from rats pretreated in vivo with phenobarbital, we observed an increase in CP‐induced malformations and embryotoxicity compared to those of embryos exposed to CP in the presence of uninduced hepatocytes. The teratogenic bioactivation of CP was inhibited in vitro by the addition of metyrapone. When similar numbers of hepatocytes were used for metabolic activation of CP the induced embryotoxicity was greater in the presence of rabbit and hamster hepatocytes than with rat hepatocytes. Development of procedures for the culture of rat embryos with hepatocytes from other species suggests the utility of this in vitro system for the investigation of species differences in sensitivity to chemical teratogens.
Microsomal mediated embryo toxicity due to superoxide radicals
Authors: Iannaccone P. M.
Published: February 2006

The transfer of toxic substances to the developing fetus with deleterious results on pregnancy outcome is well known. It is now clear that the preimplantation period of development is susceptible to such exposure. This early developmental period is a time of critical genetic determination and a period during which much membrane synthesis is occurring. Since disruption of membrane formation might be important in the disorganization of embryos, microsomal protein‐mediated toxicity in mouse blastocysts was explored. The embryos were shown to be completely disorganized by exposure to induced microsomal proteins in the presence of NADPH. All of the embryos exposed died within 18 hr. The effect was completely eliminated by boiling the microsomes or by removal of NADPH. Decreasing toxicity of the exposure could be achieved by adding butyl hydroxy toluene, an antioxidant. An amount of 0.001 mg/ml resulted in 100% viability in the presence of microsomes and cofactors after 2 hr (33% viability after 18 hr); however BHT resulted in its own toxicity at 0.025 mg/ml. Superoxide dismutase, an enzyme that removes superoxide radicals by dismutation, was completely protective with as little as 0.6 IU of enzyme added to the incubation mixture.
Quantitative analysis of the metabolism of benzo(a)pyrene by transformable C3H10T1/2CL8 mouse embryo fibroblasts
Authors: Rudo Kenneth , Ellis Scott , Bryant B. J. , Lawrence Kay , Curtis Gaynelle , Garland Helen , Nesnow Stephen
Published: February 2006

The metabolism of benzo(a)pyrene [B(a)P] to organic soluble and water soluble metabolites by transformable C3H10T1/2CL8 mouse embryo fibroblasts was studied as a function of time, B(a)P concentration, and cell density. The total formation of organic‐soluble and water‐soluble metabolites increased with incubation time from 4 to 48 h and with B(a)P concentration from 4 to 40 μM. As cell density increased, the metabolic rate decreased for organic‐soluble and water‐soluble products between 6,300 and 54,000 cells/cm2 probably due to decreases in B(a)P concentrations to values below saturation. Specific organic‐soluble metabolites identified were B(a)P‐pre‐9, 10‐diols, B(a)P‐9,10‐diol, B(a)P‐7,8‐diol, B(a)P‐3,6‐quinone, B(a)P‐3‐phenol, and B(a)P‐9‐phenol. Water‐soluble metabolites were subjected to enzymatic hydrolysis with β‐glucuronidase and aryl sulfatase to identify specific conjugated products. The sulfate conjugated metabolites identifed were B(a)P‐7,8‐diol, B(a)P‐pre‐9, 10‐diols, B(a)P‐9, 10‐diol, and B(a)P‐3, 6‐quinone. The β‐glucuronic acid metabolites identified were B(a)P‐pre‐9, 10‐diols, B(a)P‐3, 6‐quinone, and B(a)P‐3‐phenol. Patterns of metabolite formation rates are discussed as to their possible effect on morphological transformation rates in C3H10T1/2 cells with respect to incubation time and cell density.
Teratogenic effects of the fungicide dinocap in the mouse
Authors: Rogers John M. , Carver Brenda , Gray L. Earl , Gray Jacqueline A. , Kavlock Robert J.
Published: February 2006

The teratogenic potential of the fungicide dinocap was evaluated in CD‐1 mice. Pregnant mice were dosed by intubation with dinocap in corn oil on gestation days 7‐16. Doses used were 0, 5, 10, 20, 40, 80, and 120 mg/kg/day, based on day 6 weight. Dams were killed on day 18, at which time fetuses were counted, weighed, and preserved for necropsy or skeletal examination. The highest dose killed 80% of the dams dosed, while 29% of the dams in the 80 mg/kg group died during dosing. There was no dose‐related maternal mortality at lower doses. Net maternal weight gain was affected only at 80 mg/kg/day. There were no live fetuses at 120 mg/kg/day. The number of live fetuses per litter was decreased and resorptions increased at 80 mg/kg. Dose‐related decreases in gravid uterus weight and fetal weight were significant at all doses of dinocap. Cleft palate was found in fetuses at 5(1/234; 0.4%), 20(46/195; 23.6%), 40(140/185; 75.5%), and 80(63/85; 74.1%) mg/kg/day. There was a dose‐related increase in supernumerary ribs and a low frequency of exencephaly and umbilical hernia at high doses. This study shows that dinocap is teratogenic in the CD‐1 mouse at doses well below those causing maternal toxicity.
Expression of the major heat shock protein (hsp 70) family during early mouse embryo development
Authors: Hahnel Ann C. , Gifford David J. , Heikkila John J. , Schultz Gilbert A.
Published: February 2006

Stress or heat shock proteins (hsp) are synthesized by most cells in response to adverse environmental conditions. In mammalian cells, the major proteins synthesized in response to stress have relative molecular weights (Mr) in the range of 68 to 74 kilodaltons (kD) and are encoded by a small multi‐gene family collectively referred to as hsp 70 genes. In unfertilized mouse eggs, no members of the hsp 70 family appear to be synthesized under normal or stressful (heat shock) conditions. At the two‐cell stage, two proteins with Mr = 74 kD and Mr = 70 kD are expressed as a consequence of developmentally activated transcription of these hsp 70 genes. No stressinduced synthesis in response to heat shock is observed at this stage. At the eight‐cell stage, constitutive synthesis of the 70‐kD protein continues, but, as in the two‐cell embryo, no heat shock induced synthesis of a novel heat shock protein is observed. By the blastocyst stage, however, an inducible protein with Mr = 68 kD is synthesized in response to heat shock in addition to constitutive synthesis of the 70‐kD protein. The constitutively synthesized cognate proteins are coded for by a set of mRNAs about 2,000 nucleotides in length. The induced hsp 68 proteins are coded for by mRNAs of larger size (about 2,600 nucleotides). Only the smaller mRNA class is detectable on Northern blots of RNA extracted from control or heat shock cleavage stage embryos and control blastocysts. As predicted from protein synthetic studies, both classes are resolved in RNA preparations derived from heat‐shocked blastocysts.
A comparison of developmental toxicity evident at term to postnatal growth and survival using ethylene glycol monoethyl ether, ethylene glycol monobutyl ether, and ethanol
Authors: Wier Patrick J. , Lewis Steven C. , Traul Karl A.
Published: February 2006

This study was designed to compare an abbreviated evaluation of uterine contents at term (teratology probe) with a modified Chernoff‐Kavlock assay (postnatal study), [Chernoff N, Kavlock RJ: J Toxicol Environ Health 10:541–550, 1982]. Mice were intubated during gestation and were evaluated for signs of toxicity. In the teratology probe, uterine contents were examined at term. In the postnatal study, offspring were examined and weighed through day 22 postpartum.

Ethylene glycol monoethyl ether (EGEE) produced embryo lethality and malformations, and decreased fetal weight at a dose level which was not maternally toxic in the teratology probe. In the postnatal study, EGEE decreased litter size and neonatal body weight; while litter size continued to decrease beyond the neonatal period, body weights of surviving pups were not significantly different from control. Pups exposed prenatally to EGEE developed kinked tail which was not apparent in fetuses or neonates.

Maternally toxic dose levels of ethylene glycol monobutyl ether and ethanol were associated with increased embryo lethality in teratology probe studies. In postnatal studies, there were no significant effects on pup growth or survival at maternally toxic dose levels.

Preliminary conclusions regarding maternal and developmental toxicity were comparable based on the teratology probe or postnatal study. Both assays measure litter size and offspring weight, but the teratology probe measures resorption incidence which may be a more sensitive index of prenatal death than number of live born. Neither fetal weight nor neonatal weight reliably predict permanent alteration of growth. A postnatal study permits detection of internal malformations or functional defects which reduce postnatal survival and gross abnormalities which appear postnatally.
An indirect assessment of the Chernoff/Kavlock assay
Authors: Palmer A. K.
Published: February 2006

The underlying principle of the Chernoff/Kavlock assay—namely that prenatal toxicity can be detected by means of simple postnatal endpoints—is strongly supported by experience gained in performing reproductive and developmental toxicity studies to international requirements.

Compared with the supposedly more sophisticated procedures of regulatory tests, the simpler endpoints of the assay offer greater speed, economy, consistency and reliability. At the most, there is only a marginal loss of sensitivity in respect of detecting the lowest dosage at which effects may occur.

The assay is best used as a concept rather than as a protocol with, “tailored” variations providing effective means of priority selection from a series of chemicals, especially analogue series. The underlying principle is applicable at levels through and beyond formal regulatory testing.
DNA damage in the central nervous system of rats after in vivo exposure to chemical carcinogens: Correlation with the induction of brain tumors
Authors: Robbiano Luigi , Brambilla Marco
Published: February 2006

The alkaline elution technique has been used to evaluate DNA damage in brain of rats treated with a single equimolar dose of 14 carcinogens of different chemical structure. A clear‐cut increase of DNA elution rate, which is considered indicative of DNA fragmentation, was produced by 10 compounds known to induce the development of tumors in the rat central nervous system: N‐nitroso‐N‐methylurea, N‐nitroso‐N‐ethylurea, N‐nitroso‐N‐butylurea, N‐nitroso‐N‐methylurethane, methyl methanesulfonate, ethyl methanesulfonate, dimethyl sulfate, diethyl sulfate, 1,3‐propansultone, and procarbazine. Similar amounts of DNA fragmentation were produced by both potent and weak brain carcinogens. In contrast, any significant increase of DNA elution rate was absent in rats treated with N‐methyl‐N′‐nitro‐N‐nitrosoguanidine, N‐nitrosodimethylamine, N‐nitrosodiethylamine, and β‐propiolactone, all of which are devoid of carcinogenic activity for the rat central nervous system. These results suggest that the described in vivo brain DNA damage/alkaline elution assay deserves further studies on a wide number of carcinogens and noncarcinogens aimed to establish its possible usefulness for a qualitative preliminary assessment of the ability of a compound to induce neurogenic tumors.
Multiple abnormalities in the ultraviolet light response of cultured fibroblasts derived from patients with the basal cell nevus syndrome
Authors: Nagasawa H. , Burke M. J. , Little F. F. , McCone E. F. , Chan G. L. , Little J. B.
Published: February 2006

Basal Cell Nevus Syndrome (BCNS) is a rare autosomal‐dominant inherited disorder associated with a marked hypersusceptibility to spontaneous and radiation‐induced skin cancer. We examined the changes in cell survival, unscheduled DNA synthesis (UDS) and the frequency of sister chromatid exchanges (SCE) induced by ultraviolet light (UVL) in confluent normal and BCNS fibroblasts. BCNS cells appeared slightly hypersensitive to the cytotoxic effects of UVL. The rate of UDS induced by UVL exposure in normal cell strains increased linearly following doses up to 30 J/m2, whereas in BCNS cells UDS became saturated at doses of 10 J/m2 showing no further increase with doses up to 30 J/m2. UDS activity persisted for longer periods after UVL exposure in BCNS as compared with normal cells. The dose‐response relationship for UVL‐induced SCE was similar in normal and BCNS fibroblasts. However, the frequencies of UVL‐induced SCE declined to near background levels in normal cells following 12–24 hr of confluent holding prior to subculture whereas they remained elevated in BCNS cells with holding times up to 24 hr after UVL exposure. Overall, these results suggest that BCNS fibroblasts may have a diminished capacity for the repair of some type of DNA damage as compared with normal fibroblasts.
Transplacental teratogenesis and mutagenesis in mouse fetuses treated with cyclophosphamide
Authors: Porter A. J. , Singh S. M.
Published: February 2006

We studied transplacental fetotoxicity, teratogenicity, and mutagenicity in Swiss Webster mice following different doses of cyclophosphamide (CP; 0, 5, 10, 15, or 20 mg/kg), a well‐known mutagen/teratogen, on day 12 of gestation. The fetal survival and weight on day 18 of gestation decreased significantly with increasing CP dose (P < 0.01). The CP‐treated fetuses were also dysmorphic (e.g., shortened limbs, digital defects, cleft palate, open eyes, and hydrocephaly) and the percentage of dysmorphology increased with increasing CP doses (P < 0.01). To evaluate mutagenesis, a separate group of females received 5‐bromodeoxyuridine tablet (50‐mg) implants on day 12 of gestation and a CP treatment 8 h later. Fetal liver cells were harvested 24 h post‐BrdU implant to analyze sister chromatid exchange (SCE) frequency and micronuclei. CP caused a significant increase in the SCEs per fetal liver cell from 3.4 ± 0.02 (control) to 90.0 ± 0.04 (20 mg/kg CP) (P < 0.01). The increasing CP dose was also related to an increase in micronuclei. The data suggest that CP is transplacentally toxic, teratogenic, and mutagenic. Further analyses of the data suggest that the mutagenic effects of CP may in fact contribute indirectly to the CP‐related teratogenic effects. Such conclusions are based on path analysis with directional causations associated with SCEs per cell and the dysmorphic features studied.