Adverse outcome pathway (AOP): α2u-globulin nephropathy and kidney tumors in male rats.

The National Research Council's vision of using adverse outcome pathways (AOPs) as a framework to assist with toxicity assessment for regulatory requirements of chemical assessment has continued to gain traction since its release in 2007. The need to expand the AOP knowledge base has gained urgency, with the U.S. Environmental Protection Agency's directive to eliminate reliance on animal toxicity testing by 2035. To meet these needs, our goal was to elucidate the AOP for male-rat-specific kidney cancer. Male-rat-specific kidney tumors occur through the ability of structurally diverse substances to induce α2u-globulin nephropathy (α2u-N), a well-studied mode of action (MoA) not relevant in humans that results in kidney tumor formation in male rats. An accepted AOP may help facilitate the differentiation from other kidney tumors MoAs. Following identification and review of relevant in vitro and in vivo literature, both the MIE and subsequent KEs were identified. Based on the weight of evidence from the various resources, the confidence in this AOP is high. Uses of this AOP include hazard identification, development of in vitro assays to determine if the MoA is through α2u-N and not relevant to humans resulting in decreased use of animals, and regulatory applications.

RespiraTox - Development of a QSAR model to predict human respiratory irritants.

Respiratory irritation is an important human health endpoint in chemical risk assessment. There are two established modes of action of respiratory irritation, 1) sensory irritation mediated by the interaction with sensory neurons, potentially stimulating trigeminal nerve, and 2) direct tissue irritation. The aim of our research was to, develop a QSAR method to predict human respiratory irritants, and to potentially reduce the reliance on animal testing for the identification of respiratory irritants. Compounds are classified as irritating based on combined evidence from different types of toxicological data, including inhalation studies with acute and repeated exposure. The curated project database comprised 1997 organic substances, 1553 being classified as irritating and 444 as non-irritating. A comparison of machine learning approaches, including Logistic Regression (LR), Random Forests (RFs), and Gradient Boosted Decision Trees (GBTs), showed, the best classification was obtained by GBTs. The LR model resulted in an area under the curve (AUC) of 0.65, while the optimal performance for both RFs and GBTs gives an AUC of 0.71. In addition to the classification and the information on the applicability domain, the web-based tool provides a list of structurally similar analogues together with their experimental data to facilitate expert review for read-across purposes.

Environment-Sensitive Fluorescence of 7-Nitrobenz-2-oxa-1,3-diazol-4-yl (NBD)-Labeled Ligands for Serotonin Receptors.

Serotonin is a neurotransmitter that plays a crucial role in the regulation of several behavioral and cognitive functions by binding to a number of different serotonin receptors present on the cell surface. We report here the synthesis and characterization of several novel fluorescent analogs of serotonin in which the fluorescent NBD (7-nitrobenz-2-oxa-1,3-diazol-4-yl) group is covalently attached to serotonin. The fluorescent ligands compete with the serotonin1A receptor specific radiolabeled agonist for binding to the receptor. Interestingly, these fluorescent ligands display a high environmental sensitivity of their fluorescence. Importantly, the human serotonin1A receptor stably expressed in CHO-K1 cells could be specifically labeled with one of the fluorescent ligands with minimal nonspecific labeling. Interestingly, we show by spectral imaging that the NBD-labeled ligand exhibits a red edge excitation shift (REES) of 29 nm when bound to the receptor, implying that it is localized in a restricted microenvironment. Taken together, our results show that NBD-labeled serotonin analogs offer an attractive fluorescent approach for elucidating the molecular environment of the serotonin binding site in serotonin receptors. In view of the multiple roles played by the serotonergic systems in the central and peripheral nervous systems, these fluorescent ligands would be useful in future studies involving serotonin receptors.

Strain-related differences in mouse lung gene expression over a two-year period of inhalation exposure to styrene: Relevance to human risk assessment.

Both CD-1 and C57BL/6 wildtype (C57BL/6-WT) mice show equivalent short-term lung toxicity from exposures to styrene, while long-term tumor responses are greater in CD-1 mice. We analyzed lung gene expression from styrene exposures lasting from 1-day to 2-years in male mice from these two strains, including a Cyp2f2(-/-) knockout (C57BL/6-KO) and a Cyp2F1/2A13/2B6 transgenic mouse (C57BL/6-TG). With short term exposures (1-day to 1-week), CD-1 and C57BL/6-WT mice had thousands of differentially expressed genes (DEGs), consistent with changes in pathways for cell proliferation, cellular lipid metabolism, DNA-replication and inflammation. C57BL/6-WT mice responded within a single day; CD-1 mice required several days of exposure. The numbers of exposure related DEGs were greatly reduced at longer times (4-weeks to 2-years) with enrichment only for biological oxidations in C57BL/6-WT and metabolism of lipids and lipoproteins in CD-1. Gene expression results indicate a non-genotoxic, mouse specific mode of action for short-term styrene responses related to activation of nuclear receptor signaling and cell proliferation. Greater tumor susceptibility in CD-1 mice correlated with the presence of the Pas1 loci, differential Cytochrome P450 gene expression, down-regulation of Nr4a, and greater inflammatory pathway activation. Very few exposure-related responses occurred at any time in C57BL/6-KO or -TG mice indicating that neither the short term nor long term responses of styrene in mice are relevant endpoints for assessing human risks.

Based on an analysis of mode of action, styrene-induced mouse lung tumors are not a human cancer concern.

Based on 13 chronic studies, styrene exposure causes lung tumors in mice, but no tumor increases in other organs in mice or rats. Extensive research into the mode of action demonstrates the key events and human relevance. Key events are: metabolism of styrene by CYP2F2 in mouse lung club cells to ring-oxidized metabolites; changes in gene expression for metabolism of lipids and lipoproteins, cell cycle and mitotic M-M/G1 phases; cytotoxicity and mitogenesis in club cells; and progression to preneoplastic/neoplastic lesions in lung. Although styrene-7,8-oxide (SO) is a common genotoxic styrene metabolite in in vitro studies, the data clearly demonstrate that SO is not the proximate toxicant and that styrene does not induce a genotoxic mode of action. Based on complete attenuation of styrene short-term and chronic toxicity in CYP2F2 knockout mice and similar attenuation in CYP2F1 (humanized) transgenic mice, limited metabolism of styrene in human lung by CYP2F1, 2 + orders of magnitude lower SO levels in human lung compared to mouse lung, and lack of styrene-related increase in lung cancer in humans, styrene does not present a risk of cancer to humans.

Editor's Highlight: Complete Attenuation of Mouse Lung Cell Proliferation and Tumorigenicity in CYP2F2 Knockout and CYP2F1 Humanized Mice Exposed to Inhaled Styrene for up to 2 Years Supports a Lack of Human Relevance.

Styrene is a mouse-specific lung carcinogen, and short-term mode of action studies have demonstrated that cytotoxicity and/or cell proliferation, and genomic changes are dependent on CYP2F2 metabolism. The current study examined histopathology, cell proliferation, and genomic changes in CD-1, C57BL/6 (WT), CYP2F2(-/-) (KO), and CYP2F2(-/-) (CYP2F1, 2B6, 2A13-transgene) (TG; humanized) mice following exposure for up to 104 weeks to 0- or 120-ppm styrene vapor. Five mice per treatment group were sacrificed at 1, 26, 52, and 78 weeks. Additional 50 mice per treatment group were followed until death or 104 weeks of exposure. Cytotoxicity was present in the terminal bronchioles of some CD-1 and WT mice exposed to styrene, but not in KO or TG mice. Hyperplasia in the terminal bronchioles was present in CD-1 and WT mice exposed to styrene, but not in KO or TG mice. Increased cell proliferation, measured by KI-67 staining, occurred in CD-1 and WT mice exposed to styrene for 1 week, but not after 26, 52, or 78 weeks, nor in KO or TG mice. Styrene increased the incidence of bronchioloalveolar adenomas and carcinomas in CD-1 mice. No increase in lung tumors was found in WT despite clear evidence of lung toxicity, or, KO or TG mice. The absence of preneoplastic lesions and tumorigenicity in KO and TG mice indicates that mouse-specific CYP2F2 metabolism is responsible for both the short-term and chronic toxicity and tumorigenicity of styrene, and activation of styrene by CYP2F2 is a rodent MOA that is neither quantitatively or qualitatively relevant to humans.

Assessing molecular initiating events (MIEs), key events (KEs) and modulating factors (MFs) for styrene responses in mouse lungs using whole genome gene expression profiling following 1-day and multi-week exposures.

Styrene increased lung tumors in mice at chronic inhalation exposures of 20ppm and greater. MIEs, KEs and MFs were examined using gene expression in three strains of male mice (the parental C57BL/6 strain, a CYP2F2(-/-) knock out and a CYP2F2(-/-) transgenic containing human CYP2F1, 2A13 and 2B6). Exposures were for 1-day and 1, 4 and 26weeks. After 1-day exposures at 1, 5, 10, 20, 40 and 120ppm significant increases in differentially expressed genes (DEGs) occurred only in parental strain lungs where there was already an increase in DEGs at 5ppm and then many thousands of DEGs by 120ppm. Enrichment for 1-day and 1-week exposures included cell cycle, mitotic M-M/G1 phases, DNA-synthesis and metabolism of lipids and lipoproteins pathways. The numbers of DEGs decreased steadily over time with no DEGs meeting both statistical significance and fold-change criteria at 26weeks. At 4 and 26weeks, some key transcription factors (TFs) - Nr1d1, Nr1d2, Dbp, Tef, Hlf, Per3, Per2 and Bhlhe40 - were upregulated (|FC|>1.5), while others - Npas, Arntl, Nfil3, Nr4a1, Nr4a2, and Nr4a3 - were down-regulated. At all times, consistent changes in gene expression only occurred in the parental strain. Our results support a MIE for styrene of direct mitogenicity from mouse-specific CYP2F2-mediated metabolites activating Nr4a signaling. Longer-term MFs include down-regulation of Nr4a genes and shifts in both circadian clock TFs and other TFs, linking circadian clock to cellular metabolism. We found no gene expression changes indicative of cytotoxicity or activation of p53-mediated DNA-damage pathways.

Pattern of Clinical Presentation, Laboratory Findings and Mortality Risk Among Patients of Scrub Typhus in Western Himalayas.

OBJECTIVE: To study the pattern of the clinical presentation, laboratory findings and mortality risk among patients infected by scrub typhus in Western Himalayan region of India. METHODS: We studied all serologically confirmed cases of scrub typhus admitted to our hospital from July 2010 through December 2011. Presence of IgM antibodies to scrub typhus antigens by ELISA was considered as serological confirmation of the diagnosis. We observed the symptoms, signs, laboratory findings, risk factors for death from the time of admission till discharge/death. We performed bivariate and logistic regression analysis to look for independent risk factors for death. RESULTS: Total 253 patients were studied. All patients came from rural areas. More than 2/3rd were women and below 50 years of age each. High grade fever (90%), chills and rigors (68%), conjunctival suffusion (50%), body aches (43%), headache (41%), myalgias (37%), abdominal pain (21%), lymphadenopathy (17%), splenomegaly (17%), cough (16%), altered sensorium (16%), vomiting (15%), hepatomegaly (13%) were main presenting features. Eschar was present in 112 (44%) patients. Transaminitis (49%); and abnormal renal functions (31%) were predominant biochemical abnormalities. All patients were empirically treated with doxycycline/azithromycin. Thirteen (5%) patients died. CONCLUSIONS: In our region, rural women below 50 years of age presenting with high grade fever with or without eschar should be strongly suspected to be having scrub typhus. Renal dysfunction and altered sensorium are significant mortality risk factors (p < 0.05) and need to be identified at early stage to improve the treatment outcomes.

The Maryland self-referral law: history and implications.

Many previous studies have shown that nonradiologist physicians who can refer advanced MRI and CT examinations to themselves or within their practices use these modalities at a much higher rate than those who refer their examinations to unaffiliated radiology facilities. This led Maryland to pass a unique self-referral law in 1993 to directly address self-referred advanced imaging. The authors discuss the politics and economics of self-referral and provide a comprehensive review of the creation, progression, and impact of this landmark law.

Developing a chair side, safe and non-invasive procedure for assessment of blood glucose level using gingival crevicular bleeding in dental clinics.

AIM: To study the accessibility of chair side blood glucose non-invasive screening method for diabetes mellitus during routine periodontal examination. MATERIALS AND METHODS: Fifteen non-diabetics and 15 newly onset type 2 diabetics patients with moderate to severe periodontitis were selected after meeting inclusion and exclusion criteria. Periodontal pocket probing was performed using a Williams Graduated periodontal probe. Blood oozing from gingival sulcus of anterior teeth following periodontal pocket probing was collected with stick of a glucose self-monitoring device. As control, finger stick capillary blood was taken. RESULTS: A statistically significant correlation was observed between the blood glucose level of gingival crevicular blood (GCB) and peripheral fasting blood (PFB) of diabetic subjects. The mean GCB glucose level of the subjects in diabetic group was 172.27 ± 5.02 mg/dl while mean PFB glucose was 167.80 ± 8.87 mg/dl. The correlation coefficient of diabetic and non-diabetic subjects were r = +0.715 and r = +0.619, respectively. CONCLUSION: The results suggested that blood oozing during routine periodontal examination may be used for diabetic mellitus screening in a dental office setting without the need for any extra procedure.

Cellular telephone as reservoir of bacterial contamination: myth or fact.

OBJECTIVE: To assess bacterial contamination of cellular telephone of dental care personnel, and to determine factors contributing to their contamination. MATERIALS AND METHODS: A descriptive, cross-sectional study was conducted, which included 300 people using a cellular telephone The study group (hundred in each group) comprised of Dental Health Care Personnel (DHCP), In-Hospital Personnel (IHP) and Out-Hospital Personnel (OHP) of a dental college cum hospital. Swab was wiped along the front and all sides of cellular handset and it was incubated in glucose broth. The swab was subplated onto growth media plates made with half Mac Conkey's agar and half blood agar and allowed to incubate for 48 hours at 37(o)C. Isolates were tested for antimicrobial susceptibility. RESULT: The analysis of presence or absence of microorganisms in the DHCP, IHP and OHP group showed no pyogenic growth in 28%, 31% and 41% cases respectively, the distribution of which was not significant (p>.05). Among non potential pathogens, spore bearing gram positive bacilli were seen in 20 cases of DHCP group, 16 cases of IHP group and 17 cases of OHP group; the distribution of which was not significant (p>.05) Among potential pathogens, significant differences were observed in the distribution of growth of Enterobacter (p<.001), Pseudomonas species (p<.05), Acinetobacter bacteria (p<.05) and Methicillin-resistant Staphylococcus aureus (MRSA) bacteria (p<.001) between the participants of different groups. CONCLUSION: RESULTs of this study showed that fomites such as cellular telephones can potentially act as "Trojan horses", thus causing Hospital-Acquired Infections (HAIs) in the dental setting.

Scrub typhus and leptospirosis co-infection in Himalayan region.

Scrub typhus and leptospirosis are both zoonosis and systemic febrile illnesses with diverse clinical manifestations and they may present with similar signs and symptoms. We present a case of co-infection of scrub typhus and leptospirosis from Himachal Pradesh in the Himalayan region of India.

Scrub typhus presenting as acute abdomen.

Scrub typhus, a zoonosis, is a systemic febrile illness with diverse clinical manifestations. It may also present with signs and symptoms of acute abdomen. We present two serologically confirmed cases of scrub typhus presenting with acute abdomen that were managed conservatively with antibiotics.

Fluid tagging for CT colonography: effectiveness of a 2-hour iodinated oral preparation after incomplete optical colonoscopy.

OBJECTIVE: To evaluate the distal extent and attenuation of bowel opacification achieved after administration of a single low volume dose of oral contrast 2 hours before computed tomographic colonography (CTC) after incomplete optical colonoscopy. METHODS: This retrospective study included 144 patients undergoing CTC after incomplete colonoscopy from April 2006 to July 2008 at 2 separate medical centers. Each patient received 20 to 30 mL of diatrizoate meglumine and diatrizoate sodium solution 2 hours before being scanned. RESULTS: The distalmost extent of opacification was: stomach/small bowel, n = 13; cecum, n = 2; ascending colon, n = 7; transverse colon, n = 19; descending colon, n = 14; sigmoid colon, n = 24; rectum, n = 65. The mean attenuation of each opacified segment was: cecum, 449 Hounsfield units (HU); ascending colon, 474 HU; transverse colon, 468 HU; descending colon, 421 HU; sigmoid colon, 391 HU; and rectum, 382 HU. In 103 (71.5%) patients, oral contrast reached the distal colon (descending colon, sigmoid colon, or rectum). The oral contrast did not reach the colon in only 13 (9.0%) patients. CONCLUSIONS: Oral administration of a small volume hyperosmolar oral contrast agent 2 hours before CTC results in satisfactory colonic opacification in the majority of patients. Adding same-day fluid tagging in incomplete colonoscopy patients presenting for completion CTC should result in adequate fluid opacification for most of the colon, especially proximal segments not visualized at the time of incomplete colonoscopy.

Hormone receptor status of breast cancer in the himalayan region of northern India.

The role of hormone receptors as a prognostic and therapeutic tool in breast cancer is widely accepted. The frequency of nonreactivity of estrogen and progesterone receptors in breast cancer patients of India is much more common than in the West. This study was conducted with the aim of analysis of steroid receptor status in breast cancer with clinico-pathological characteristics from the northern hilly state of Himachal Pradesh, India located in the region of the Western Himalayas. Fifty five consecutive patients with the diagnosis of breast cancer were included in this study. Detailed clinical and histopathologic data was recorded in all cases. Estrogen receptor and progesterone receptor status was evaluated by immunohistochemistry. Chi-square test was used for statistical analysis. On immunohistochemical staining, 34.5% cases proved to be estrogen receptor positive and 36.4% cases progesterone receptor positive. The results in the present study documented low estrogen receptor and progesterone receptor positivity in breast cancer from this region of India.

A comparative study of Barron's rubber band ligation with Kshar Sutra ligation in hemorrhoids.

Despite a long medical history of identification and treatment, hemorrhoids still pose a challenge to the medical fraternity in terms of finding satisfactory cure of the disease. In this study, Kshar Sutra Ligation (KSL), a modality of treatment described in Ayurveda, was compared with Barron's Rubber Band Ligation (RBL) for grade II and grade III hemorrhoids. This study was conducted in 20 adult patients of either sex with grade II and grade III hemorrhoids at two different hospitals. Patients were randomly allotted to two groups of 10 patients each. Group I patients underwent RBL, whereas patients of group II underwent KSL. Guggul-based Apamarga Kshar Sutra was prepared according to the principles laid down in ancient Ayurvedic texts and methodology standardized by IIIM, Jammu and CDRI, Lucknow. Comparative assessment of RBL and KSL was done according to 16 criteria. Although the two procedures were compared on 15 criteria, treatment outcome of grade II and grade III hemorrhoids was decided chiefly on the basis of patient satisfaction index (subjective criterion) and ability of each procedure to deal with prolapse of internal hemorrhoidal masses (objective criterion): Findings in each case were recorded over a follow-up of four weeks (postoperative days 1, 3, 7, 15 and 30). Statistical analysis was done using Student's t test for parametric data and Chi square test & Mann-Whitney test for non-parametric data. P < 0.05 was considered significant. RBL had the advantages of being an OPD procedure requiring no anesthesia and was attended by significantly lesser postoperative recumbency (P < 0.001 ) and significantly lesser pain (P < 0.005 on day 1) as compared to KSL. However, Group II (KSL) scored better in terms of treatment outcome. In Group II, there was significantly high (P < 0.05) patient satisfaction index as compared to Group I. Group II reported 100% 'cure' (absence of hemorrhoidal masses even on proctoscopy) of internal hemorrhoidal prolapse as against 80% in Group I (RBL); however, this difference was statistically insignificant (P > 0.05). Both the groups were comparable statistically on all other grounds. Kshar Sutra Ligation is a useful form of treatment for Grades II and III internal hemorrhoids.

Pattern of cutaneous manifestations in diabetes mellitus.

BACKGROUND: Diabetes mellitus affects individuals of all ages and socioeconomic status. Skin is affected by the acute metabolic derangements as well as by chronic degenerative complications of diabetes. AIMS: To evaluate the prevalence of skin manifestations in patients with diabetes mellitus. To analyze the prevalence and pattern of skin disorders among diabetic patients from this region of Western Himalayas. MATERIALS AND METHODS: One hundred consecutive patients with the diagnosis of diabetes mellitus and having skin lesions, either attending the diabetic clinic or admitted in medical wards were included in this study. RESULTS: The common skin disorders were: Xerosis (44%), diabetic dermopathy (36%), skin tags (32%), cutaneous infections (31%), and seborrheic keratosis (30%). CONCLUSION: Skin is involved in diabetes quite often and the manifestations are numerous. High prevalence of xerosis in our diabetic population is perhaps due to cold and dry climatic conditions in the region for most of the time in the year.

Ceramide kinase regulates phospholipase C and phosphatidylinositol 4, 5, bisphosphate in phototransduction.

Phosphoinositide-specific phospholipase C (PLC) is a central effector for many biological responses regulated by G-protein-coupled receptors including Drosophila phototransduction where light sensitive channels are activated downstream of NORPA, a PLCbeta homolog. Here we show that the sphingolipid biosynthetic enzyme, ceramide kinase, is a novel regulator of PLC signaling and photoreceptor homeostasis. A mutation in ceramide kinase specifically leads to proteolysis of NORPA, consequent loss of PLC activity, and failure in light signal transduction. The mutant photoreceptors also undergo activity-dependent degeneration. Furthermore, we show that a significant increase in ceramide, resulting from lack of ceramide kinase, perturbs the membrane microenvironment of phosphatidylinositol 4, 5, bisphosphate (PIP(2)), altering its distribution. Fluorescence image correlation spectroscopic studies on model membranes suggest that an increase in ceramide decreases clustering of PIP(2) and its partitioning into ordered membrane domains. Thus ceramide kinase-mediated maintenance of ceramide level is important for the local regulation of PIP(2) and PLC during phototransduction.

Automated multidetector computed tomography evaluation of subacutely infarcted myocardium.

BACKGROUND: Delayed enhanced (DE) multidetector computed tomography (MDCT) can identify acute and chronic myocardial infarct. To our knowledge, automated techniques for infarct quantification on DE-MDCT have not been used. OBJECTIVE: We evaluated an automated signal intensity (SI) threshold method for quantification of subacute myocardial infarct and identified and quantified microvascular obstruction (MO) in subacute infarct. METHODS: DE-MDCT imaging was performed on 5 pigs 6-7 days after mid left anterior descending artery occlusion-reperfusion. DE-MDCT images were compared with triphenyl tetrazolium chloride (TTC) staining for infarct quantification and with hematoxylin and eosin (H&E) staining for MO quantification. Pixels with SI more than the mean SI of a remote normal myocardial region (SI(remote)) plus 2 times the standard deviation (SI(remote) + 2 SD) value were considered infarct pixels. The ratio of infarct to total area of a given slice, the percentage of infarct area per slice (PIS), was calculated. MO as a percentage of total infarct area was also calculated. RESULTS: The average density values on DE-MDCT (5 minutes after contrast injection) were remote normal myocardium of 93 +/- 19 Hounsfield units (HU), infarct myocardium of 159 +/- 40 HU, blood of 140 +/- 26 HU, and MO of 85 +/- 30 HU. PIS(MDCT) showed substantial agreement with PIS(TTC) (y = 1.003x + 4.12; R = 0.90, P < 0.05). A relation was also shown between MO determined by MDCT compared with H&E staining (y = 0.74x + 3.4). CONCLUSIONS: We show the feasibility of using a semiautomated SI threshold technique for quantification of subacute myocardial infarct. We also show the persistent MO in subacute myocardial infarct on DE-MDCT images.

Role of tryptophan residues in gramicidin channel organization and function.

The linear peptide gramicidin forms prototypical ion channels specific for monovalent cations and has been used extensively to study the organization, dynamics, and function of membrane-spanning channels. The tryptophan residues in gramicidin channels are crucial for maintaining the structure and function of the channel. We explored the structural basis for the reduction in channel conductance in the case of single-tryptophan analogs of gramicidin with three Trp --> hydrophobic substitutions using a combination of fluorescence approaches, which include red edge excitation shift and membrane penetration depth analysis, size-exclusion chromatography, and circular dichroism spectroscopy. We show here that the gramicidin analogs containing single-tryptophan residues adopt a mixture of nonchannel and channel conformations, as evident from analysis of membrane penetration depth, size-exclusion chromatography, and backbone circular dichroism data. These results are potentially useful in analyzing the effect of tryptophan substitution on the functioning of other ion channels and membrane proteins.