Toxicity testing in the 21st century: progress in the past decade and future perspectives.

Advances in the biological sciences have led to an ongoing paradigm shift in toxicity testing based on expanded application of high-throughput in vitro screening and in silico methods to assess potential health risks of environmental agents. This review examines progress on the vision for toxicity testing elaborated by the US National Research Council (NRC) during the decade that has passed since the 2007 NRC report on Toxicity Testing in the 21st Century (TT21C). Concomitant advances in exposure assessment, including computational approaches and high-throughput exposomics, are also documented. A vision for the next generation of risk science, incorporating risk assessment methodologies suitable for the analysis of new toxicological and exposure data, resulting in human exposure guidelines is described. Case study prototypes indicating how these new approaches to toxicity testing, exposure measurement, and risk assessment are beginning to be applied in practice are presented. Overall, progress on the 20-year transition plan laid out by the US NRC in 2007 has been substantial. Importantly, government agencies within the United States and internationally are beginning to incorporate the new approach methodologies envisaged in the original TT21C vision into regulatory practice. Future perspectives on the continued evolution of toxicity testing to strengthen regulatory risk assessment are provided.

BK Polyomavirus Genomic Integration and Large T Antigen Expression: Evolving Paradigms in Human Oncogenesis.

Human polyomaviruses are ubiquitous, with primary infections that typically occur during childhood and subsequent latency that may last a lifetime. Polyomavirus-mediated disease has been described in immunocompromised patients; its relationship to oncogenesis is poorly understood. We present deep sequencing data from a high-grade BK virus-associated tumor expressing large T antigen. The carcinoma arose in a kidney allograft 6 years after transplantation. We identified a novel genotype 1a BK polyomavirus, called Chapel Hill BK polyomavirus 2 (CH-2), that was integrated into the BRE gene in chromosome 2 of tumor cells. At the chromosomal integration site, viral break points were found, disrupting late BK gene sequences encoding capsid proteins VP1 and VP2/3. Immunohistochemistry and in situ hybridization studies demonstrated that the integrated BK virus was replication incompetent. We propose that the BK virus CH-2 was integrated into the human genome as a concatemer, resulting in alterations of feedback loops and overexpression of large T antigen. Collectively, these findings support the emerging understanding that viral integration is a nearly ubiquitous feature in polyomavirus-associated malignancy and that unregulated large T antigen expression drives a proliferative state that is conducive to oncogenesis. Based on the current observations, we present an updated model of polyomavirus-mediated oncogenesis.

Loss of ovarian estrogens causes only mild deterioration of glucose homeostasis in female ZDF rats preventable by voluntary running exercise.

Studies on normoglycemic ovariectomized Sprague-Dawley rats have provided insights about the effects of estrogen deficiency on insulin resistance in lean individuals. It is not completely clear if subjects with pre-established obesity and insulin resistance are at greater risk of developing type 2 diabetes when ovarian estrogens are no longer secreted, and if physical activity can protect against this susceptibility. Contrasting with their male counterparts, obese and insulin resistant female ZDF (Zucker diabetic fatty) rats do not become hyperglycemic when fed a standard diet. The aim of the study was to evaluate the hypothesis that withdrawal of ovarian estrogens in insulin resistant female ZDF rats would trigger overt hyperglycemia, provided they remain physically inactive. Female ZDF rats underwent either an ovariectomy (OVX) or a simulated surgery (SHAM). Thereafter, OVX rats engaged either in voluntary wheel cage running (OVX-Active), or like the Sham rats, remained sedentary (OVX-Sed) for 6 weeks. Fasting glycemia, insulinemia, and glucose tolerance were not altered in OVX-Sed as compared to SHAM-Sed rats. However, OVX-Sed rats showed altered liver triglyceride and glycogen contents, increased pancreatic insulin content and reduced insulin-stimulated muscle pAKT as compared to SHAM-Sed rats. Physical activity in OVX rats lowered fasting glucose and insulin levels, improved glucose tolerance and insulin-stimulated skeletal muscle glucose uptake as compared to OVX-Sed rats. OVX-induced alterations in pancreatic insulin content and liver glycogen and triglyceride contents were significantly improved by physical activity. Loss of ovarian estrogens did not cause overt hyperglycemia in insulin-resistant female ZDF rats. Physical activity improved glucose homeostasis despite estrogen deficiency.

Liver fat accumulation may be dissociated from adiposity gain in ovariectomized rats.

OBJECTIVE: The purpose of the present study was to establish a model of rats prone and resistant to intra-abdominal fat accumulation in response to ovariectomy (Ovx-P and Ovx-R) and to determine its relationship with molecular biomarkers. DESIGN: Two experiments were conducted in which female rats were either sham-operated (Sham) or ovariectomized (Ovx). In the first experiment, ovariectomized rats were stratified into three tertiles based on intra-abdominal adipose tissue mass. To strengthen the Ovx-P/Ovx-R model, we conducted a second experiment in which the numbers of rats in each group were extended and in which different molecular markers were measured. At the end of a 6-8-week period, ovariectomized rats that displayed the lower abdominal fat accumulation (lower tertile) were labelled as Ovx-R and those in the upper tertile as Ovx-P. RESULTS: Ovx-R rats displayed similar abdominal fat gain to Sham rats whereas Ovx-P rats depicted abdominal fat mass twice as high as that of Sham and Ovx-R rats. Despite the difference in abdominal adiposity, liver fat content was ~50% higher (p < 0.01) in both Ovx-R and Ovx-P rats compared to Sham rats. In addition, both Ovx-R and Ovx-P rats depicted higher HOMA-IR scores (p < 0.05) and lower (p < 0.01) hepatic gene expression of leptin receptor-b and -e, microsomal transfer protein (MTP), and diacylglycerol acyltransferase-2 (DGAT-2) compared to Sham rats. CONCLUSION: The present findings indicate that estrogen withdrawal-induced hepatic steatosis and associated insulin resistance may be dissociated from abdominal fat accumulation and suggest that a decrease in leptin action through a down-regulation of leptin receptors and a decrease in very low density lipoprotein production through a down-regulation of MTP and DGAT-2 may be factors responsible for this observation in the absence of peripheral fat gain.

Development and characterization of a monoclonal antibody against Taura syndrome virus.

We produced a panel of monoclonal antibodies (MAbs) from the fusion of Taura syndrome virus variants from Belize (TSV-BZ) immunized BALB/cJ mouse spleen cells and non-immunoglobulin secreting SP2/0 mouse myeloma cells. One antibody, 2C4, showed strong specificity and sensitivity for TSV in dot-blot immunoassay and immunohistochemistry (IHC) analysis. The MAb reacted against native TSV-BZ, TSV variants from Sinaloa, Mexico (TSV-SI) and TSV variants from Hawaii (TSV-HI) in dot-blot immunoassay. By IHC, the antibody identified the virus in a pattern similar to the digoxigenin-labelled TSV-cDNA probe for the TSV-BZ, TSV-HI and TSV-SI variants, but not for the TSV variants from Venezuela (TSV-VE) and the TSV variants from Thailand (TSV-TH). MAb 2C4 did not react against other shrimp pathogens or with normal shrimp tissue. Western blot analysis showed a strong reaction against CP2, a region of high antigenic variability amongst TSV variants. This antibody has potential diagnostic application in detection and differentiation of certain TSV biotypes.

A simple clinico-histopathological composite scoring system is highly predictive of graft outcomes in marginal donors.

The predictive value of pre-implantation biopsies versus clinical scores has not been studied extensively in marginal donors. Pre-implantation biopsies were performed in 313 kidneys from donors that were > or = 50 years of age (training set, n = 191; validation set, n = 122). The value of the donor clinical parameters and histological results in predicting 1-year estimated glomerular filtration rate (eGFR) <25 mL/min/1.73 m(2) was retrospectively evaluated. In multivariate analysis, the only clinical parameters associated with low eGFR were donor hypertension and a serum creatinine level > or =150 micromol/L before organ recovery. Clinical scores (Nyberg and Pessione) were not significantly associated with graft function. Regarding histological parameters, univariate analysis showed that glomerulosclerosis (GS) (p = 0.02), arteriolar hyalinosis (p = 0.03) and the Pirani (p = 0.02) and chronic allograft damage index (CADI) (p = 0.04) histological scores were associated with low eGFR. The highest performance in predicting low eGFR was achieved using a composite score that included donor serum creatinine (> or =150 micromol/L or <150 micromol/L), donor hypertension and GS (> or =10% or <10%). The validation set confirmed the critical importance of taking into account biopsy and clinical parameters during marginal donor evaluation. In conclusion, clinical scores are weak predictors of graft outcomes with marginal donors. Instead, a simple and convenient composite score strongly predicts graft function and survival and may facilitate optimal allocation of marginal donors.

[Submental myocutaneous island flaps: anatomical study and prospective use]. / Le lambeau musculo-cutané sous mental en îlot: étude anatomique et perspectives d'utilisation.

The authors describe a myocutaneous cervical island flap based on the submental vessels. The anatomy based on 12 fresh cadaver dissection is outlined the submental arteries were injected with methylene blue, and the flap design and technique were studied. The flap has a long, reliable pedicle and cutaneous dimension measured 10 x 5 cm. The flap has an excellent skin color match and wide are of rotation and can extend to the whole homolateral face, the whole oral cavity, the whole homolateral oropharyngeal. The donor site scare dissimulated under the mandible is perfectly acceptable.

Impact of heterogeneity in the predictive value of kinetic parameters in canine osteosarcoma.

Intratumoral heterogeneity has been identified as a potential problem in the efficacy of predictive assays. Canine osteosarcoma is an extremely heterogeneous solid tumor that has been shown to be an excellent model for the human disease. Intratumoral heterogeneity of kinetic parameters and the effect of heterogeneity on predicting outcome of treatment (time until metastasis) were studied in dogs with naturally occurring osteosarcoma. Dogs were treated with amputation or tumor excision and limb-sparing followed by chemotherapy with cisplatin. Kinetic parameters evaluated included v, duration of DNA synthesis (Ts), and potential doubling time (Tpot), determined using in vivo labeling with bromodeoxyuridine and flow cytometry. In 30 tumors, multiple samples were obtained and evaluated. There was significantly more variation between tumors from different dogs than intratumoral variation of v, Ts, and Tpo. Variations in v, Ts, and Tpot within a tumor were associated with both sample location and tumor subpopulation. Time to metastasis was determined in 51 dogs with tumors sampled for kinetics. Multiple samples were available from 25 of these tumors. Cox proportional hazard analysis was performed using either the fastest or slowest Tpot from each sample. The fastest available Tpots were highly significant (P < 0.001) for prediction of outcome. The slowest available Tpots were also significant predictors, although the statistical strength was compromised (P = 0.024). Obtaining at least two samples in large tumors known to be heterogeneous is recommended to improve the predictive ability of Tpot. v is a more limited predictor but can useful when Tpot is not available. In canine osteosarcoma, an extremely heterogeneous tumor, kinetic parameters were shown to be predictors of outcome.

Cancer risk assessment of 1,3-butadiene.

This paper discusses the Environmental Protection Agency's (EPA) risk assessment of 1,3-butadiene. The assessment focuses on estimation of increased cancer risk to populations living near industrial sources of 1,3-butadiene emissions rather than occupationally exposed populations. Incremental cancer risk estimates based on extrapolation from laboratory animal data are presented. Pharmacokinetic data published since the EPA's 1985 assessment are incorporated, which somewhat alters the earlier assessment of cancer risk. Characterization of emission sources, estimates of ambient air concentrations, and population exposure are also discussed. The estimate presented in this paper of excess cancer cases resulting from point source exposure to 1,3-butadiene is decreased to approximately 40% of the estimate published in 1985 from 6.4 in 10 to 2.5 chances in 10 for a lifetime exposure to 1 ppm. The current estimate is no more than eight additional cancer incidences in the general population. Increased risk to the most exposed individuals is not anticipated to be greater than 1 in 10. This reduction in the risk estimate is due to a change in the estimate of 1,3-butadiene potency (i.e., incremental unit risk estimate) based on incorporation of new pharmacokinetic data.

In vitro alkylation of calf thymus DNA by acrylonitrile. Isolation of cyanoethyl-adducts of guanine and thymine and carboxyethyl-adducts of adenine and cytosine.

Reaction of the rodent carcinogen acrylonitrile (AN) at pH 5.0 and/or pH 7.0 for 10 and/or 40 days with 2'-deoxyadenosine (dAdo), 2'-deoxycytidine (dCyd), 2'-deoxyguanosine (dGuo), 2'-deoxyinosine (dIno), N6-methyl-2'-deoxyadenosine (N6-Me-dAdo) and thymidine (dThd) resulted in the formation of cyanoethyl and carboxyethyl adducts. Adducts were not detected after 4 h. The adducts isolated were 1-(2-carboxyethyl)-dAdo (1-CE-dAdo), N6-CE-dAdo, 3-CE-dCyd, 7-(2-cyanoethyl)-Gua (7-CNE-Gua), 7,9-bis-CNE-Gua, imidazole ring-opened 7,9-bis-CNE-Gua, 1-CNE-dIno, 1-CE-N6-Me-dAdo and 3-CNE-dThd. Structures were assigned on the basis of UV spectra and electron impact (EI), chemical ionization (CI), desorption chemical ionization (DCI) and Californium-252 fission fragment ionization mass spectra. Evidence is presented which strongly suggests that N6-CE-dAdo was formed by Dimroth rearrangement of 1-CE-dAdo during the reaction between AN and dAdo. The carboxyethyl adducts resulted from initial cyanoethylation (by Michael addition) at a ring nitrogen adjacent to an exocyclic nitrogen atom followed by rapid hydrolysis of the nitrile moiety to a carboxylic acid. It was postulated that the facile hydrolysis is an autocatalyzed reaction resulting from the formation of a cyclic intermediate between nitrile carbon and exocyclic nitrogen. AN was reacted with calf thymus DNA (pH 7.0, 37 degrees C, 40 days) and the relative amounts of adducts isolated were 1-CE-Ade (26%), N6-CE-Ade (8%), 3-CE-Cyt (1%), 7-CNE-Gua (26%), 7,9-bis-CNE-Gua (4%), imidazole ring-opened 7,9-bis-CNE-Gua (19%) and 3-CNE-Thy (16%). Thus a carcinogen once adducted to a base in DNA was shown to be subsequently modified resulting in a mixed pattern of cyanoethylated and carboxyethylated AN-DNA adducts. Three of the adducts (1-CE-Ade, N6-CE-Ade and 3-CE-Cyt) were identical to adducts previously reported by us to be formed following in vitro reaction of the carcinogen beta-propiolactone (BPL) and calf thymus DNA. The results demonstrate that AN can directly alkylate DNA in vitro at a physiological pH and temperature.

Effects of acrylonitrile on tissue glutathione concentrations in rat, mouse, and hamster.

Glutathione (GSH) is implicated in acrylonitrile (ACN) detoxification. To add to the understanding of ACN detoxification we studied the effects of ACN on GSH levels in various tissues and species. GSH in four tissues (brain, lung, liver, and kidney) from three species (rat, mouse and hamster) were assessed at five time points subsequent to ACN treatment. ACN-induced decreases in GSH were most pronounced in the rat and occurred in all four tissues and were less pronounced in mouse and hamster tissues. Liver GSH was most effected in all species.