Toxicol Pathol Research Articles

A Technical Guide to Sampling the Beagle Dog Nervous System for General Toxicity and Neurotoxicity Studies.

Beagle dogs are a key nonrodent species in nonclinical safety evaluation of new biomedical products. The Society of Toxicologic Pathology (STP) has published "best practices" recommendations for nervous system sampling in nonrodents during general toxicity studies (Toxicol Pathol 41[7]: 1028-1048, 2013), but their adaptation to the Beagle dog has not been defined specifically. Here we provide 2 trimming schemes suitable for evaluating the unique neuroanatomic features of the dog brain in nonclinical toxicity studies. The first scheme is intended for general toxicity studies (Tier 1) to screen test articles with unknown or no anticipated neurotoxic potential; this plan using at least 7 coronal hemisections matches the STP "best practices" recommendations. The second trimming scheme for neurotoxicity studies (Tier 2) uses up to 14 coronal levels to investigate test articles where the brain is a suspected or known target organ. Collection of spinal cord, ganglia (somatic and autonomic), and nerves for dogs during nonclinical studies should follow published STP "best practices" recommendations for sampling the central (Toxicol Pathol 41[7]: 1028-1048, 2013) and peripheral (Toxicol Pathol 46[4]: 372-402, 2018) nervous systems. This technical guide also demonstrates the locations and approaches to collecting uncommonly sampled peripheral nervous system sites.

New aspects in deriving health-based guidance values for bromate in swimming pool water

Bromate, classified as a EU CLP 1B carcinogen, is a typical by-product of the disinfection of drinking and swimming pool water. The aim of this study was (a) to provide data on the occurrence of bromate in pool water, (b) to re-evaluate the carcinogenic MOA of bromate in the light of existing data, (c) to assess the possible exposure to bromate via swimming pool water and (d) to inform the derivation of cancer risk-related bromate concentrations in swimming pool water. Measurements from monitoring analysis of 229 samples showed bromate concentrations in seawater pools up to 34 mg/L. A comprehensive non-systematic literature search was done and the quality of the studies on genotoxicity and carcinogenicity was assessed by Klimisch criteria (Klimisch et al., Regul Toxicol Pharmacol 25:1–5, 1997) and SciRAP tool (Beronius et al., J Appl Toxicol, 38:1460–1470, 2018) respectively. Benchmark dose (BMD) modeling was performed using the modeling average mode in BMDS 3.1 and PROAST 66.40, 67 and 69 (human cancer BMDL10; EFSA 2017). For exposure assessment, data from a wide range of sources were evaluated for their reliability. Different target groups (infants/toddlers, children and adults) and exposure scenarios (recreational, sport-active swimmers, top athletes) were considered for oral, inhalation and dermal exposure. Exposure was calculated according to the frequency of swimming events and duration in water. For illustration, cancer risk-related bromate concentrations in pool water were calculated for different target groups, taking into account their exposure using the hBMDL10 and a cancer risk of 1 in 100,000. Convincing evidence was obtained from a multitude of studies that bromate induces oxidative DNA damage and acts as a clastogen in vitro and in vivo. Since statistical modeling of the available genotoxicity data is compatible with both linear as well as non-linear dose–response relationships, bromate should be conservatively considered to be a non-threshold carcinogen. BMD modeling with model averaging for renal cancer studies (Kurokawa et al., J Natl. Cancer Inst, 1983 and 1986a; DeAngelo et al., Toxicol Pathol 26:587–594, 1998) resulted in a median hBMDL10 of 0.65 mg bromate/kg body weight (bw) per day. Evaluation of different age and activity groups revealed that top athletes had the highest exposure, followed by sport-active children, sport-active adults, infants and toddlers, children and adults. The predominant route of exposure was oral (73–98%) by swallowing water, followed by the dermal route (2–27%), while the inhalation route was insignificant (< 0.5%). Accepting the same risk level for all population groups resulted in different guidance values due to the large variation in exposure. For example, for an additional risk of 1 in 100,000, the bromate concentrations would range between 0.011 for top athletes, 0.015 for sport-active children and 2.1 mg/L for adults. In conclusion, the present study shows that health risks due to bromate exposure by swimming pool water cannot be excluded and that large differences in risk exist depending on the individual swimming habits and water concentrations.

Nervous System Sampling for General Toxicity and Neurotoxicity Studies in the Laboratory Minipig With Emphasis on the Göttingen Minipig.

The use of minipigs as an alternative nonclinical species has increased in the last 20 years. The Society of Toxicologic Pathology (STP) has produced generic "best practice" recommendations for nervous system sampling in nonrodents during general toxicity studies (Toxicol Pathol 41[7]: 1028-1048, 2013), but their adaptation to the minipig has not been attempted. Here, we describe 2 trimming schemes suitable for evaluating the unique neuroanatomic features of the minipig brain in nonclinical toxicity studies. The first scheme is intended for general toxicity studies (Tier 1) to screen agents with unknown or no anticipated neurotoxic potential; this approach using 7 coronal hemisections accords with the published STP "best practice" recommendations. The second trimming scheme for neurotoxicity studies (Tier 2) uses 14 coronal hemisections and 2 full coronal sections to investigate toxicants where the nervous system is a suspected or known target organ. Collection of spinal cord, ganglia (somatic and autonomic), and nerves from minipigs during nonclinical studies should follow published STP "best practice" recommendations for sampling the central (CNS, Toxicol Pathol 41[7]: 1028-1048, 2013) and peripheral (PNS, Toxicol Pathol 46[4]: 372-402, 2018) nervous systems.

Acetaminophen and the Developing Lung: Could There Be Lifelong Consequences?

Acetaminophen and the Developing Lung: Could There Be Lifelong Consequences?

Nervous System Sampling for General Toxicity and Neurotoxicity Studies in Rabbits.

Although manuscripts for multiple species recommending nervous system sampling for histopathology evaluation in safety assessment have been published in the past 15 years, none have addressed the laboratory rabbit. Here, we describe 2 trimming schemes for evaluating the rabbit brain in nonclinical toxicity studies. In both schemes, the intact brain is cut in the coronal plane to permit bilateral assessment. The first scheme is recommended for general toxicity studies (tier 1) in screening agents where there is no anticipated neurotoxic potential; this 6-section approach is consistent with the Society of Toxicologic Pathology (STP) "best practice" recommendations for brain sampling in nonrodents (Toxicol Pathol 41: 1028-1048, 20131). The second trimming scheme is intended for dedicated neurotoxicity studies (tier 2) to characterize known or suspected neurotoxicants where the nervous system is a key target organ. This tier 2 strategy relies on coronal trimming of the whole brain into 3-mm-thick slices and then evaluating 12 sections. Collection of spinal cord, ganglia, and nerve specimens for rabbits during nonclinical studies should follow published STP "best practice" recommendations for sampling the central nervous system1 and peripheral nervous system (Toxicol Pathol 46: 372-402, 20182).

Proliferative and Nonproliferative Lesions of the Rat and Mouse Central and Peripheral Nervous Systems: New and Revised INHAND Terms.

Harmonization of diagnostic terminology used during the histopathologic analysis of rodent tissue sections from nonclinical toxicity studies will improve the consistency of data sets produced by laboratories located around the world. The INHAND Project (International Harmonization of Nomenclature and Diagnostic Criteria for Lesions in Rats and Mice) is a cooperative enterprise of 4 major societies of toxicologic pathology to develop a globally accepted standard vocabulary for proliferative and nonproliferative lesions in rodents. A prior manuscript (Toxicol Pathol 2012;40[4 Suppl]:87S-157S) defined multiple diagnostic terms for toxicant-induced lesions, common spontaneous and age-related changes, and principal confounding artifacts in the rat and mouse central nervous system (CNS) and peripheral nervous system (PNS). The current article defines 9 new diagnostic terms and updates 2 previous terms for findings in the rodent CNS and PNS, the need for which has become evident in the years since the publication of the initial INHAND nomenclature for findings in rodent neural tissues. The nomenclature presented in this document is also available electronically on the Internet at the goRENI website (http://www.goreni.org/).

Effect of dexmedetomidine on ischemia-reperfusion injury of liver and kidney tissues in experimental diabetes and hepatic ischemia-reperfusion injury induced rats

Background: Reperfusion following ischemia can lead to more injuries than ischemia itself especially in diabetic patients. The aim of this study was to evaluate the effect of dexmedetomidine on ischemia-reperfusion injury (IRI) in rats with have hepatic IRI and diabetes mellitus.

Transmigration of Leukocytes Across Epithelial Monolayers.

Migration of leukocytes through epithelial monolayers represents an essential step in the generation of an inflammatory response and is often seen in inflammatory conditions such as Crohn's disease (Matthews et al., Toxicol Pathol 42:91-98, 2014) and asthma (Lambrecht and Hammad, Nat Med 18:684-692, 2012). Transepithelial migration involves adhesion to the basal surface of the epithelium before migration through the epithelial cell layer to the apical surface. Analyzing this process can present a technical challenge due to complications of using a coculture model and trying to recapitulate an intact monolayer. Here we describe two methods of assessing transepithelial migration based on a Transwell assay, the first of which measures the apical-basal migration of epithelial cells and the second "Inverted" transwell assay that measures basal-apical transmigration of leukocytes and therefore more closely mimics the in vivo process.

Bayesian Semiparametric Model for Pathway-Based Analysis with Zero-Inflated Clinical Outcomes

In this paper, we propose a semiparametric regression approach for identifying pathways related to zero-inflated clinical outcomes, where a pathway is a gene set derived from prior biological knowledge. Our approach is developed by using a Bayesian hierarchical framework. We model the pathway effect nonparametrically into a zero-inflated Poisson hierarchical regression model with an unknown link function. Nonparametric pathway effect was estimated via a kernel machine, and the unknown link function was estimated by transforming a mixture of the beta cumulative density function. Our approach provides flexible nonparametric settings to describe the complicated association between gene expressions and zero-inflated clinical outcomes. The Metropolis-within-Gibbs sampling algorithm and Bayes factor were adopted to make statistical inferences. Our simulation results support that our semiparametric approach is more accurate and flexible than zero-inflated Poisson regression with the canonical link function, which is especially true when the number of genes is large. The usefulness of our approach is demonstrated through its applications to the Canine data set from Enerson et al. (Toxicol Pathol 34:27–32, 2006). Our approach can also be applied to other settings where a large number of highly correlated predictors are present.

Abstract 4580: Expression of γH2AX as a biomarker of genotoxic carcinogen in the urinary bladder of rats

Abstract Background: Bladder cancer is the seventh most common cancer in males worldwide. Recent studies have demonstrated that DNA double-strand breaks are recognized to be a serious injury leading to genome instability and cancer development. The rapid phosphorylation of histone H2AX at Serine 139, to become γH2AX, occurs in response to DNA double-strand breaks. Roles of γH2AX in carcinogenesis are still unclear, and there are few reports on the association with bladder cancer. We have previously demonstrated that γH2AX expression is induced in urinary bladders of N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN)-treated rat (Toyoda et al., J Toxicol Pathol, 26: 215-221, 2013). To evaluate the value of γH2AX as a biomarker for carcinogenicity and genotoxicity to the urinary bladder, we performed an immunohistochemical analysis of γH2AX expression in chemical-treated rats. Methods: Six-week-old male F344 rats were treated with 0.05% BBN, 1.8% 2-nitroanisole (2-NA), 2% 2,2-bis (bromomethyl)-1,3-propanediol (BMP), 0.1% phenethyl isothiocyanate (PEITC), 3% melamine, or 3% uracil in either drinking water (BBN) or diet (other chemicals) for 4 weeks. Animals were sacrificed at 4 or 6 weeks of the experiment, and immunohistochemistry for γH2AX in urinary bladders was performed. Results: At week 4, γH2AX expression in the urothelium were significantly increased in BBN and 2-NA groups compared with the controls. γH2AX expression ratio (positive cells/1000 cells) in BBN, 2-NA and control groups were 76±21, 111±37 and 0.8±0.6, respectively. γH2AX was recognized as dot-like foci in nuclei of epithelial cells, and the positive cells were mainly located near the basal layer of the mucosa. In BMP and PEITC groups, γH2AX expression was mainly observed in umbrella cells and intermediate cells, respectively. In melamine and uracil groups, there were diffuse urothelial hyperplasia and focal expression of γH2AX especially in the basal layer. The expression ratios of γH2AX in BMP, PEITC, melamine and uracil groups were 8.3±6.6, 26±9.0, 16±11 and 11±11, respectively. Two weeks after withdrawal of administration, although the expression ratio of γH2AX was markedly reduced in all groups, it remained high in BBN (17±1.9) and 2-NA (19±3.4) groups compared with the controls (0.6±0.9). Discussion and conclusion: BBN and 2-NA, genotoxic bladder carcinogens, induced more extensive γH2AX expression in the urothelium than other non-genotoxic (melamine and uracil) or non-carcinogenic chemicals (BMP and PEITC), and γH2AX-positive cells remained in relatively large numbers even 2 weeks after withdrawal of its administration. In addition, ratio of basal cells, intermediate cells and umbrella cells in γH2AX-positive cells showed similar patterns in BBN/2-NA and melamine/uracil groups. These data indicate that γH2AX has potential as a biomarker for early detection of genotoxic carcinogen in the urinary bladder. Citation Format: Takeshi Toyoda, Young-Man Cho, Jun-ichi Akagi, Yasuko Mizuta, Kumiko Ogawa. Expression of γH2AX as a biomarker of genotoxic carcinogen in the urinary bladder of rats. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4580. doi:10.1158/1538-7445.AM2015-4580

BPA as a Mammary Carcinogen: Early Findings Reported in Rats

Perinatal exposure of rodents to low doses of bisphenol A (BPA) has been associated with altered mammary gland development and increased propensity for mammary tumors later in life.1 A study by Tufts University researchers in the current issue of EHP suggests that BPA may act as a malignant mammary gland carcinogen in rats at internal doses comparable to those seen in humans.2 Prenatal exposures to chemicals can yield different results than studies in adult animals.3 Previous studies have shown that rats and mice exposed prenatally to both BPA and a known carcinogen in adulthood grew more tumors faster than animals exposed only to the carcinogen.4 In a 2007 study, senior author Ana Soto and colleagues detected intraductal hyperplasias—overgrowths of cells considered to be precursors to mammary gland carcinomas in both rodents and humans—at postnatal day 50 and 95 in Wistar Furth rats exposed prenatally to environmentally relevant levels of BPA.5 Some of these animals formed ductal carcinoma in situ (DCIS)—localized tumors that sometimes but not always progress to malignancy. According to the authors, the current study is the first to report malignant tumor formation following developmental exposure to environmentally relevant levels of BPA. Soto, a cancer researcher at the Tufts University School of Medicine, calls the findings unexpected. “We were measuring response to internal dose and looking for preneoplastic lesions. Instead, what we saw were full-blown tumors,” she says. Soto and colleagues treated pregnant Sprague-Dawley rats with varying amounts of BPA during gestation and lactation or during gestation only. They measured levels of total BPA and unconjugated or free BPA in the blood of pregnant dams and their fetuses and nursing pups. Unconjugated BPA in blood is considered the hormonally active form of the chemical. They detected total and unconjugated BPA in all the treated dams and their fetuses and in one-third of the nursing pups exposed to 250 µg BPA per kg body weight per day via subcutaneous pump. Exposure of dams to a BPA dose of 250 µg/kg BW/day either during gestation or during gestation and lactation resulted in an average blood level of unconjugated BPA of 1.25 ng/mL. (By comparison, a 2010 review of human biomonitoring studies reported mean blood levels of BPA in healthy adults of approximately 1 ng/mL.6) The researchers observed six mammary gland tumors in female offspring exposed perinatally to BPA at doses ranging from 0.25 to 250 µg/kg BW/day as early as 90 days after birth. Five of the tumors were classified as adenocarcinomas—malignant tumors with the potential to be invasive and metastatic. No malignant tumors were detected in any of the control animals. However, because there were so many more exposed rats than controls (230 versus 65), the investigators cannot rule out the possibility that the observed tumors developed spontaneously, despite prior evidence that female Sprague-Dawley rats rarely form spontaneous malignant tumors before 210 days of age.7,8 Figure 1 Micrographs show lesions detected in mammary gland tissue from female rats exposed to varying doses of BPA either during gestation only or during gestation and lactation. The findings suggest BPA may be a malignant mammary carcinogen, but the study must ... Epidemiological studies have suggested that exposure to elevated estrogen levels in the womb may be associated with an increase in a woman’s lifetime risk of breast cancer.9,10 Exposure to the estrogen mimic BPA is widespread, with one report from the nationally representative National Health and Nutrition Examination Survey (NHANES) finding that 90% of respondents over age 6 years had detectable levels of BPA in their urine.11 (NHANES, like most biomonitoring studies, measures only urinary levels of total and free BPA, not blood levels.12) “From the point of view of human health research, the most notable aspect of the study is that the blood levels are very comparable to those found in humans, which isn’t always the case with animal studies,” says epidemiologist Barbara Cohn, director of the Oakland, California–based Child Health and Development Studies, a cohort of more than 15,000 mothers, daughters, and granddaughters aimed at deciphering the role that environmental exposures play in the development of diseases such as breast cancer.13 “This finding may be of great public health significance,” Cohn says. “But without a question it must be replicated before we can know for sure whether BPA is a complete carcinogen.” She says information on the impact of developmental exposure to BPA on breast cancer in humans will also be critical for understanding the public health significance of the findings.

Proliferative and non-proliferative lesions of the rat and mouse integument.

The INHAND (International Harmonization of Nomenclature and Diagnostic Criteria for Lesions in Rats and Mice) project is a joint initiative of the societies of toxicological pathology from Europe (ESTP), Great Britain (BSTP), Japan (JSTP) and North America (STP). Its aim is to develop an internationally-accepted nomenclature for proliferative and non-proliferative lesions in laboratory rodents. A widely accepted international harmonization of nomenclature in laboratory animals will decrease confusion among regulatory and scientific research organizations in different countries and will provide a common language to increase and enrich international exchanges of information among toxicologists and pathologists. The purpose of this publication is to provide a standardized nomenclature for classifying microscopical lesions observed in the integument of laboratory rats and mice. Example colour images are provided for most lesions. The standardized nomenclature presented in this document and additional colour images are also available electronically at http://www.goreni.org. The nomenclature presented herein is based on histopathology databases from government, academia, and industrial laboratories throughout the world, and covers lesions that develop spontaneously as well as those induced by exposure to various test materials. (DOI: 10.1293/tox.26.27S; J Toxicol Pathol 2013; 26: 27S–57S)

Proliferative and non-proliferative lesions of the rat and mouse soft tissue, skeletal muscle and mesothelium.

The INHAND Project (International Harmonization of Nomenclature and Diagnostic Criteria for Lesions in Rats and Mice) is a joint initiative of the Societies of Toxicologic Pathology from Europe (ESTP), Great Britain (BSTP), Japan (JSTP), and North America (STP) to develop an internationally accepted nomenclature for proliferative and nonproliferative lesions in laboratory animals. The purpose of this publication is to provide a standardized nomenclature for classifying lesions observed in the soft tissues including skeletal muscle as well as the mesothelium of rats and mice. The standardized nomenclature of lesions presented in this document is also available electronically on the Internet (http://www.goreni.org/). Sources of material included histopathology databases from government, academia, and industrial laboratories throughout the world. Content includes spontaneous developmental and aging lesions as well as those induced by exposure to test materials. A widely accepted and utilized international harmonization of nomenclature for lesions in soft tissues, skeletal muscle and mesothelium in laboratory animals will decrease confusion among regulatory and scientific research organizations in different countries and provide a common language to increase and enrich international exchanges of information among toxicologists and pathologists. (DOI: 10.1293/tox.26.1S; J Toxicol Pathol 2013; 26: 1S–26S)

Long-term Pulmonary Responses to Quadweekly Intermittent Intratracheal Spray Instillations of Magnetite (Fe&lt;sub&gt;3&lt;/sub&gt;O&lt;sub&gt;4&lt;/sub&gt;) Nanoparticles for 52 Weeks in Fischer 344 Rats

Information about potential risks of iron nanomaterials is still limited, while a wide variety of applications are expected. We recently reported acute phase responses of male and female Fischer 344 rats after a single intratracheal spray instillation of Fe3O4 nanoparticles (magnetite), clearly showing dose-dependent pulmonary inflammatory changes (Tada et al., J Toxicol Pathol 25, 233–239, 2012). The present study assessed long-term responses of male and female Fischer 344 rats to multiple administrations of magnetite. Ten-week-old male and female Fischer 344 rats (n=20/group) were exposed to a total of 13 quadweekly intermittent intratracheal spray instillations of magnetite during the experimental period of 52 weeks, at doses of 0, 0.2 (low), 1.0 (medium) and 5.0 (high-dose) mg/kg body weight per administration. Absolute and relative lung weights of the high-dose group were significantly higher than those of the control group. Macroscopically, slight enlargement and scattered black patches were recognized in the lungs and the lung-associated lymph nodes of the high-dose group. Histopathologically, infiltration of macrophages phagocytosing magnetite (all dose groups) and of chronic inflammatory cells (medium- and high-dose males and high-dose females), alveolar bronchiolization and granuloma (high-dose group) were observed. In addition, alveolar hyperplasias were observed in some rats of the high-dose group, and cytoplasmic overexpression of β-catenin protein was immunohistochemically found in such lesions. The present results clearly show that instilled magnetite causes chronic inflammatory responses in the lung. These responses occur in a dose-dependent manner without apparent differences among sexes

Malformations of the Great Vessels in the Neonatal Rat Induced by N-(2-Aminoethyl)ethanolamine

The reproductive and developmental toxicity of aminoethylethanolamine was evaluated in a standard screening study (OECD, 1995: Organisation for economic co-operation and development. Paris, France), in which groups of Wistar rats (10/sex/group) were administered the test substance by gavage at dosage levels of 50, 250, or 1000 mg/kg/day (groups 2-4, respectively). A control group received the vehicle, doubly distilled water. No live pups were delivered in group 4, and there was a higher incidence of stillborn offspring and reduced postnatal survival in group 3. Macroscopic changes in groups 2 and 3 were primarily related to the great vessels and characterized by dilations, aneurysms, and altered course of the aorta, pulmonary trunk, carotids, and the ductus arteriosus. A follow-up study was conducted to characterize the low dose-response, using dosage levels of 0, 0.2, 1, 5, or 50 mg/kg/day (groups 1-5, respectively). Given the expected scarcity of the lesions in control offspring, each group consisted of 25 animals of each sex. Macroscopic examination revealed a high incidence (18.5%) of aneurysm-bearing offspring in group 5 litters, and single offspring (0.3-0.4%) with aneurysms in groups 3 and 4. Microscopic examination revealed dissecting aneurysms in offspring from all aminoethylethanolamine treatment groups, without a clear dose-response between groups 2 and 4 (0.6%, 1.2%, and 0.3%, respectively), and focal hemorrhages in all groups including the control. In comparison, the background incidence of aneurysms in untreated 4-day old offspring was 0.2% (Treumann etal., 2011: Toxicol Pathol 39:969-974). Consequently, the findings in groups 2-4 cannot be conclusively attributed to treatment.

Introduction to Hepatic Drug Metabolizing Enzyme Induction in Drug Safety Evaluation Studies

The following three articles represent the output of a combined effort initiated by the Scientific Regulatory Policy Committee of the Society of Toxicologic Pathology to provide a unified review of current scientific practices and relevant literature and provide suggestions regarding the recognition, interpretation, and risk assessment of hepatic drug metabolizing enzyme (DME) induction studies. The core objective was to provide a review that the scientific community including pathologists, regulatory scientists, toxicologists, investigative scientists, and others would find valuable for managing, designing, and interpreting toxicity studies supporting regulatory filings. Three working groups composed of scientists from industry, academia, and regulatory agencies were convened to review the available literature on important aspects of the interpretation and risk assessment of hepatic microsomal DME enzyme induction in three publications. The three reviews are as follows: "Effects of Hepatic Drug Metabolizing Enzyme Induction on Clinical Pathology Parameters in Animals and Man," Toxicol Pathol "Hepatic Drug Metabolizing Enzyme Induction: Microscopic and Ultrastructural Appearance," Toxicol Pathol "Hepatic Drug Metabolizing Enzyme Induction and Implications for Preclinical and Clinical Risk Assessment," Toxicol Pathol The purpose of this introduction is not to summarize the articles but rather to frame the series and to provide a common mechanistic introduction.

Cytoskeletal staining in rectal cancer: problems

Dear Editor: I read the recent publication “Cytokeratin staining for complete remission in rectal cancer after chemoradiation” by Gonullu et al. (Int J Colorectal Dis. 2010; 25: 805–809) with great interest. Gonullu et al. concluded that “cytokeratin IHC offers a means of improving staging accuracy and thus provides useful information for prognosis and treatment decisions for patients with rectal cancer who had a clinical CR after CRT.” The problems of the techniques “falsenegative and/or false-positive results (Painter et al. Toxicol Patho. 2010; 38: 131–141)” should be mentioned. In a recent study on immostaining of sentinel lymph node mapping in colorectal cancer, false negative seems to be very clinically important (Sommariva et al. Eur J Surg Oncol. 2010;36:130–134). A mucinous histology seem to interfere with the reliability of staging (Sommariva et al. Eur J Surg Oncol. 2010;36:130–134). Nevertheless, the reported sensitivity and specificity of the cytokeratin staining in diagnosis of malignancy are about 80% and 75%, which cannot be considered good (Golijianin et al. J Urol. 2000; 164: 1922–1925). Many factors including tissue type, fixation time, quality control on specimen collection and frozen sectioning, and antibody specificity are mentioned for their impact on the result of IHC (Painter et al. Toxicol Pathol. 2010; 38: 131–141). How to solve the basic diagnostic problems and control of quality of the whole IHC process is required before implementation of the staining technique for real usage

STP Debate on the Desirability of an International Mechanism for Recognizing Qualified Toxicologic Pathologists

The June 2009 Town Hall meeting of the Society of Toxicologic Pathology (STP) and a subsequent survey considered whether or not STP should endorse a published proposal (Toxicol Pathol 37: 553-561, 2009) by the International Federation of Societies of Toxicologic Pathologists (IFSTP) to provide global recognition by credential review for toxicologic pathologists engaged in regulatory-type, nonclinical toxicology studies. One-third (374 of 1082) of STP members answered the survey. The majority of respondents rejected the IFSTP proposal (55% against) but favored the concept of global recognition (57% for), if available to both anatomic pathologists and clinical pathologists (67% for). Members preferred recognition by credential review (49% for) or via an internationally authored "best practices" document detailing the ideal educational and work experiences required for entry-level proficiency in toxicologic pathology (43% for). Therefore, the STP Executive Committee does not endorse the current IFSTP proposal but will continue discussions on global recognition of qualified toxicologic pathologists with other societies of toxicologic pathology.

Modern cross‐linking strategies for synthesizing acellular hemoglobin‐based oxygen carriers

Unmodified cell-free hemoglobin (Hb) is structurally unstable when transfused into the blood stream (Valeri et al., Artif Cells Blood Substit Immobil Biotechnol. 2000;28:451-475; Chan et al., Toxicol Pathol. 2000;28:635-642; Eike, Dissertation, 2005; Eike and Palmer, Biotechnol Prog. 2004;20:946-952). This review examines some of the latest chemical strategies used over the last 5 years to intra- and intermolecularly cross-link Hb, thereby stabilizing its quaternary structure. Therefore, this work will address the following aspects: (1) site-specific chemical modifications of Hb and (2) non-site-specific chemical modifications of Hb, including, but not limited to, PolyHeme, Hemopure, Oxyglobin, and SOD-Hb. Current strategies for synthesizing PEGylated Hb is outside the scope of this review and will not be discussed herein. For a more thorough review of PEGylated Hb, the reader is directed to the following works: Cabrales and Friedman, Transfus Alternatives in Transfus Med. 2007;9:281-293 and Winslow, Biochim Biophys Acta, 2008;1784(10):1382-1386.

Immunohistochemical Localization of RPA-1; Comment on Paper by Zhang et al., Toxicol Pathol 36;397

Immunohistochemical Localization of RPA-1; Comment on Paper by Zhang et al., <i>Toxicol Pathol</i> 36;397