Cells Of Stomach Research Articles

Reprogrammed Stomach Tissue as a Renewable Source of Functional β Cells for Blood Glucose Regulation

The gastrointestinal (GI) epithelium is a highly regenerative tissue with the potential to provide a renewable source of insulin(+) cells after undergoing cellular reprogramming. Here, we show that cells of the antral stomach have a previously unappreciated propensity for conversion into functional insulin-secreting cells. Native antral endocrine cells share a surprising degree of transcriptional similarity with pancreatic β cells, and expression of β cell reprogramming factors in vivo converts antral cells efficiently into insulin(+) cells with close molecular and functional similarity to β cells. Induced GI insulin(+) cells can suppress hyperglycemia in a diabetic mouse model for at least 6 months and regenerate rapidly after ablation. Reprogramming of antral stomach cells assembled into bioengineered mini-organs in vitro yielded transplantable units that also suppressed hyperglycemia in diabetic mice, highlighting the potential for development of engineered stomach tissues as a renewable source of functional β cells for glycemic control.

Regulation of α-Transducin and α-Gustducin Expression by a High Protein Diet in the Pig Gastrointestinal Tract.

BackgroundThe expression of taste receptors (TASRs) and their signalling molecules in the gastrointestinal (GI) epithelial cells, including enteroendocrine cells (EECs), suggests they participate in chemosensing mechanisms influencing GI physiology via the release of endocrine messengers. TASRs mediate gustatory signalling by interacting with different transducers, including α-gustducin (Gαgust) and α-transducin (Gαtran) G protein subunits. This study tested whether Gαtran and Gαgust immunoreactive (-IR) cells are affected by a short-term (3 days) and long-term (30 days) high protein (Hp) diet in the pig GI tract.ResultIn the stomach, Gαgust and Gαtran-IR cells contained serotonin (5-HT) and ghrelin (GHR), while in the small and large intestine, Gαgust and Gαtran-IR colocalized with 5-HT-, cholecystokinin (CCK)- and peptide YY (PYY)-IR. There was a significant increase in the density of Gαtran-IR cells in the pyloric mucosa in both short- and long-term Hp diet groups (Hp3 and Hp30) vs. the control group (Ctr) (P<0.05), while the increase of Gαgust-IR cells in the pyloric mucosa was significant in Hp30 group vs. Ctr and vs. Hp3 (P<0.05); these cells included Gαtran / 5HT-IR and Gαtran / GHR-IR cells (P<0.05 and P<0.001 vs. Ctr, respectively) as well as Gαgust /5-HT-IR or Gαgust / GHR-IR cells (P<0.05 and P<0.01 vs. Ctr, respectively). In the small intestine, we recorded a significant increase in Gαtran-IR cells in the duodenal crypts and a significant increase of Gαgust-IR cells in the jejunal crypts in Hp3 group compared to HP30 (P<0.05). With regard to the number of Gαtran-Gαgust IR cells colocalized with CCK or 5-HT, there was only a significant increase of Gαtran / CCK-IR cells in Hp3 group compared to Ctr (P = 0.01).ConclusionThis study showed an upregulation of selected subpopulations of Gαgust / Gαtran-IR cells in distinct regions of the pig GI tract by short- and long-term Hp diet lending support to TASR-mediated effects in metabolic homeostasis and satiety mechanisms.

Invasive mouse gastric adenocarcinomas arising from Lgr5+ stem cells are dependent on crosstalk between the Hedgehog/GLI2 and mTOR pathways.

Gastric adenocarcinoma is the third most common cause of cancer-related death worldwide. Here we report a novel, highly-penetrant mouse model of invasive gastric cancer arising from deregulated Hedgehog/Gli2 signaling targeted to Lgr5-expressing stem cells in adult stomach. Tumor development progressed rapidly: three weeks after inducing the Hh pathway oncogene GLI2A, 65% of mice harbored in situ gastric cancer, and an additional 23% of mice had locally invasive tumors. Advanced mouse gastric tumors had multiple features in common with human gastric adenocarcinomas, including characteristic histological changes, expression of RNA and protein markers, and the presence of major inflammatory and stromal cell populations. A subset of tumor cells underwent epithelial-mesenchymal transition, likely mediated by focal activation of canonical Wnt signaling and Snail1 induction. Strikingly, mTOR pathway activation, based on pS6 expression, was robustly activated in mouse gastric adenocarcinomas from the earliest stages of tumor development, and treatment with rapamycin impaired tumor growth. GLI2A-expressing epithelial cells were detected transiently in intestine, which also contains Lgr5+ stem cells, but they did not give rise to epithelial tumors in this organ. These findings establish that deregulated activation of Hedgehog/Gli2 signaling in Lgr5-expressing stem cells is sufficient to drive gastric adenocarcinoma development in mice, identify a critical requirement for mTOR signaling in the pathogenesis of these tumors, and underscore the importance of tissue context in defining stem cell responsiveness to oncogenic stimuli.

The zinc-finger transcription factor SALL4 is frequently expressed in human cancers: association with clinical outcome in squamous cell carcinoma but not in adenocarcinoma of the esophagus.

SALL4 is a transcription factor originally identified as a homeotic gene essential for organ development. Early studies suggested that SALL4 is a useful marker to identify testicular and ovarian germ cell tumors. The aim of the study was to evaluate the diagnostic potential of SALL4 immunohistochemistry. Immunohistochemical staining was performed on a tissue microarray (TMA) with 3966 samples from 94 different tumor types and on a further TMA with 492 esophagus carcinomas. SALL4 immunostaining was by far most prevalent and most intensive in testicular tumors with a positivity rate of 93.1% in seminomas, 80% in mixed germ cell tumors (embryonic carcinomas/yolk sac tumors), and 18.5% in teratomas, respectively. However, SALL4 expression is not specific to germ cell tumors. We observed SALL4 positivity in non-germ cell tumors as carcinomas of the kidney (28.9% of chromophobe, 34.4% of clear cell carcinoma), in intestinal type adenocarcinoma of the stomach (10.9%), in adenocarcinoma (10.5%) and squamous cell carcinoma (7.2%) of the esophagus, and in malignant melanoma (8.1%) and invasive urothelial bladder carcinoma (20%). SALL4 expression was not found in lymphomas, in soft tissue tumors or breast tumors. At analysis of esophagus carcinoma TMA, no significant association was seen between SALL4 expression and overall survival in adenocarcinoma. However, SALL4 expression was strongly associated with worse overall survival in squamous cell carcinoma. SALL4 expression can be found at relevant frequencies in various tumors of different primary sites. SALL4 expression in squamous cell carcinoma of the esophagus may constitute a sign of dedifferentiation leading to poor patient prognosis.

In Silico Study of miRNA Based Gene Regulation, Involved in Solid Cancer, by the Assistance of Argonaute Protein.

Solid tumor is generally observed in tissues of epithelial or endothelial cells of lung, breast, prostate, pancreases, colorectal, stomach, and bladder, where several genes transcription is regulated by the microRNAs (miRNAs). Argonaute (AGO) protein is a family of protein which assists in miRNAs to bind with mRNAs of the target genes. Hence, study of the binding mechanism between AGO protein and miRNAs, and also with miRNAs-mRNAs duplex is crucial for understanding the RNA silencing mechanism. In the current work, 64 genes and 23 miRNAs have been selected from literatures, whose deregulation is well established in seven types of solid cancer like lung, breast, prostate, pancreases, colorectal, stomach, and bladder cancer. In silico study reveals, miRNAs namely, miR-106a, miR-21, and miR-29b-2 have a strong binding affinity towards PTEN, TGFBR2, and VEGFA genes, respectively, suggested as important factors in RNA silencing mechanism. Furthermore, interaction between AGO protein (PDB ID-3F73, chain A) with selected miRNAs and with miRNAs-mRNAs duplex were studied computationally to understand their binding at molecular level. The residual interaction and hydrogen bonding are inspected in Discovery Studio 3.5 suites. The current investigation throws light on understanding miRNAs based gene silencing mechanism in solid cancer.

Drug Interactions of H2 Receptor Antagonists-Ranitidine: A Review

Ranitidine has OTC drug status as well as it is an essential drug by WHO, hence it is popularly selling drug from community and hospital pharmacy. Many prescription and nonprescription drugs were found having drug interaction with ranitidine which may lead to either drug toxicity or therapy failure. This review compiles potential drug interactions of ranitidine with several categories of drugs along with effective measures. Ranitidine is in a group of drugs called histamine-2 blockers. H2 receptor antagonists continue to be prescribed usually for acid –peptic disease. Their safety record finally led to their availability as “Over the Counter” drugs. Histamine is a naturally-occurring chemical that stimulates cells in the stomach (parietal cells) to produce acid. H2-blockers inhibit the action of histamine on the cells, thus reducing the production of acid by the stomach. Ranitidine hydrochloride is prescribed for conditions such as gastroesophageal reflux disease (GERD), ulcers, Zollinger-Ellison syndrome, erosive esophagitis, upper gastrointestinal bleeding, heartburn, and other conditions where reduction of gastric secretion and acid output is desirable.

High-fat diet increases ghrelin-expressing cells in stomach, contributing to obesity

ObjectivesMechanisms of high-fat diet (HFD)–induced obesity may involve ghrelin, an orexigenic and adipogenic hormone secreted by the stomach. Previous studies showed that obese subjects may display higher numbers of ghrelin-producing cells and increased affinity of plasma immunoglobulins (Ig) for ghrelin, protecting it from degradation. The aim of this study was to determine if a HFD in mice would increase the number of ghrelin-expressing cells and affinity of ghrelin-reactive IgG. MethodsObesity in mice was induced by consumption of a 13-wk HFD. The number of preproghrelin mRNA-expressing cells in the stomach was analyzed by in situ hybridization and compared with chow-fed, nonobese controls and with genetically obese ob/ob mice. Affinity of ghrelin-reactive IgG was analyzed using surface plasmon resonance. Plasma levels of ghrelin and des-acyl ghrelin were measured. ResultsHFD resulted in 30% of body fat content versus only 8% in controls (P < 0.001). The number of preproghrelin mRNA-producing cells was 15% (P < 0.05) higher in HFD-fed mice than in controls, contrasting with ob/ob mice, having a 41% (P < 0.001) decrease. Both models of obesity had normal plasma levels of ghrelin but a decrease of its des-acylated form. Ghrelin-reactive IgG affinity was found in the micromolar range with mean values of the dissociation equilibrium constant 1.5-fold (P < 0.05) lower in HFD-fed versus control mice. ConclusionResults from the present study showed that HFD in mice induces obesogenic changes, including increased numbers of ghrelin precursor-expressing cells and increased affinity of ghrelin-reactive IgG. Such changes may contribute to the mechanisms of HFD-induced obesity.

Polysaccharides as Bacterial Antiadhesive Agents and "Smart" Constituents for Improved Drug Delivery Systems Against Helicobacter pylori Infection.

The standard eradication treatment of the hostile Helicobacter pylori (H. pylori) stomach infection is facing increasing alarming antibiotic resistance worldwide and calls for alternative strategies to the use of antibiotics. One new perspective in this direction is cytoprotective compounds for targeted prevention of the adhesion of the bacteria to the stomach host cell and to inhibit the bacterial cell-cell communication via quorum sensing by specific inhibitors. Bacterial adhesion of H. pylori to the host cells is mainly mediated by carbohydrate-protein interactions. Therefore, the use of polyvalent carbohydrates, (e.g. plant-derived polysaccharides), as potential antiadhesive compounds, seems to be a promising tool to prevent the initial docking of the bacterium to the stomach cells. Polysaccharides are common constituents of daily food, either as starch or as dietary fiber and often also function as excipients for galenic drug-delivery formulations. In addition, polysaccharides with defined pharmacodynamics action against bacterial outer membrane proteins can have potential as therapeutic tools in the treatment of bacterial infections. Some polysaccharides are known to possess antibacterial properties against gram-positive bacteria, others to inhibit bacterial colonization by blocking specific carbohydrate receptors involved in host-bacteria interaction. This mode of action is advocated as alternative antiadhesion therapy. Ongoing research is also seeking for polysaccharide-based nanoformulations with potential for local drug delivery at the stomach as novel H. pylori therapies. These approaches pose challenges concerned with the stability of the nanomaterials in the harsh conditions of the gastric environment and their capacity to adhere to the stomach mucosa. In a global scenario, geographical diversity and social habits, namely lifestyle and dietary factors, influence the prevalence of the H. pylori-associated diseases and their severity. In this context, the exploration of the biological activity of plant-derived products or polysaccharides commonly present in foods is increasingly becoming more and more attractive. This review aims to present the current state-of-the-art on the antiadhesive capacity of different polysaccharide families, on polysaccharide-based nanosystems and the proof-of-concept evidence of their potential use as alternative medicines against H. pylori.

Cover Photograph: Immunodetection of β integrin‐LP‐positive cells (green) in stomach and muscle (red) and ciliary systems (magenta) in mussel larva Mytilus trossulus (See Dyachuk et al., pp. 515–528).

Development, Growth & DifferentiationVolume 57, Issue 7 p. i-i Cover ImageFree Access Cover Photograph: Immunodetection of β integrin-LP-positive cells (green) in stomach and muscle (red) and ciliary systems (magenta) in mussel larva Mytilus trossulus (See Dyachuk et al., pp. 515–528). First published: 04 September 2015 https://doi.org/10.1111/dgd.12173AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume57, Issue7September 2015Pages i-i RelatedInformation

IMUNOLOCALIZAÇÃO DE ENTEROGLUCAGON EM CÉLULAS ENDÓCRINAS PRESENTES NO ESTÔMAGO DO MUÇUÃ &lt;italic&gt;Kinosternon scorpioides&lt;/italic&gt; (REPTILIA, CHELONIA, KINOSTERNIDAE)

&lt;title&gt;Resumo:&lt;/title&gt;&lt;p&gt;O muçuã ou jurará (&lt;italic&gt;Kinosternon scorpioides&lt;/italic&gt;) é um quelônio de pequeno porte de água doce, nativo das varzéas da região amazônica. Essa espécie é importante fonte de alimentação e renda e vem sendo estudada por apresentar vulnerabilidade em consequência da caça indiscriminada, queimadas e desmatamento e, por esta razão, tem sido fonte de pesquisa para o fornecimento de dados para a preservação da espécie. Ainda não é totalmente compreendida a atividade imunohistoquímica das células endócrinas presentes no estômago do muçuã. Sendo assim, este trabalho tem por objetivo identificar a presença do hormônio enteroglucagon e classificar as células endócrinas do estômago de muçuã. Os fragmentos de estômago foram submetidos à técnica de Hematoxilina-Eosina e da Estreptovidina peroxidase para coloração e detecção do antígeno, respectivamente. Foram encontradas células imunorreativas ao enteroglucagon em uma das três porções gástricas (cárdica, fúndica e pilórica); contudo, nas duas primeiras regiões, a imunoreatividade foi mais evidente do que na última. As células endócrinas do estômago de muçuã foram classificadas em argirófilas e argentafins e encontradas entre as células que compõem as glândulas gástricas, sendo classificadas como do "tipo aberta" e do "tipo fechada". Não houve diferença bioquímica e imunohistoquímica entre o enteroglucagon do &lt;italic&gt;K. scorpioides&lt;/italic&gt; com as de outras espécies animais.&lt;/p&gt;

To complete its replication cycle, a shrimp virus changes the population of long chain fatty acids during infection via the PI3K-Akt-mTOR-HIF1α pathway

White spot syndrome virus (WSSV), the causative agent of white spot disease (WSD), is a serious and aggressive shrimp viral pathogen with a worldwide distribution. At the genome replication stage (12 hpi), WSSV induces a metabolic rerouting known as the invertebrate Warburg effect, which boosts the availability of energy and biosynthetic building blocks in the host cell. Here we show that unlike the lipogenesis that is seen in cancer cells that are undergoing the Warburg effect, at 12 hpi, all of the long chain fatty acids (LCFAs) were significantly decreased in the stomach cells of WSSV-infected shrimp. By means of this non-selective WSSV-induced lipolysis, the LCFAs were apparently diverted into β-oxidation and used to replenish the TCA cycle. Conversely, at 24 hpi, when the Warburg effect had ceased, most of the LCFAs were significantly up-regulated and the composition was also significantly altered. In crayfish these changes were in a direction that appeared to favor the formation of WSSV virion particles. We also found that, at 24 hpi, but not at 12 hpi, the PI3K-Akt-mTOR-HIF1α pathway induced the expression of fatty acid synthase (FAS), an enzyme which catalyzes the conversion of acetyl-CoA into LCFAs. WSSV virion formation was impaired in the presence of the FAS inhibitor C75, although viral gene and viral DNA levels were unaffected. WSSV therefore appears to use the PI3K-Akt-mTOR pathway to induce lipid biosynthesis at 24 hpi in order to support viral morphogenesis.

Ink4a/Arf-Dependent Loss of Parietal Cells Induced by Oxidative Stress Promotes CD44-Dependent Gastric Tumorigenesis.

Loss of parietal cells initiates the development of spasmolytic polypeptide-expressing metaplasia (SPEM), a precancerous lesion in stomach. CD44 variant (CD44v) that enhances the ability to defend against reactive oxygen species (ROS) in epithelial cells is expressed de novo in SPEM of K19-Wnt1/C2mE mice, a transgenic model of gastric tumorigenesis, and is required for the efficient development of SPEM and gastric tumor in these animals. The role of ROS and its downstream signaling in CD44-dependent gastric tumorigenesis has remained unknown, however. With the use of the K19-Wnt1/C2mE mouse, we now show that parietal cells in the inflamed stomach are highly sensitive to oxidative stress and manifest activation of p38(MAPK) signaling by ROS. Oral treatment with the antioxidant ascorbic acid or genetic ablation of the Ink4a/Arf locus, a major downstream target of ROS-p38(MAPK) signaling, inhibited parietal cell loss and the subsequent gastric tumorigenesis. Our results indicate that signaling activated by oxidative stress in parietal cells plays a key role in CD44-dependent gastric tumorigenesis. .

CD4(+) Foxp3(+) regulatory T-cell number increases in the gastric tissue of C57BL/6 mice infected with Helicobacter pylori.

Helicobacter pylori (H. pylori), one of the most common infections, is associated with various clinical outcomes. In addition to inducing inflammation, immunological clearance of the pathogen is often incomplete. Regulatory T cells (Treg cells) have been recently demonstrated to play an important role in H. pylori infection and the final clinical outcome. The aim of this study was to investigate the number and localization of CD4(+) Foxp3(+) Treg cells in stomachs and spleens of H. pylori-infected mice. The expression levels of Foxp3 as well as anti- and pro-inflammatory cytokines before and after H. pylori triple eradication therapy were examined. We found that the percentages of CD4(+) Foxp3(+) Treg cells out of the lamina propria lymphocytes (LPLs) and spleen lymphocytes in the infection group were higher than the PBS negative control group and the treatment group. H. pylori antigen stimulation was associated with an increased number of Treg cells in vitro. Furthermore, compared with the PBS and treatment groups, a higher mRNA expression level of Foxp3 in the gastric tissue was detected in the infection group. IL-10 and TGF-β1 contents were increased significantly in the culture supernatant of spleen lymphocyte stimulated with H. pylori antigen. A marked elevation in serum IFN-γ level was observed in H. pylori-infected mice. In addition, gastric tissues of the infection group contained more Foxp3(+) cells. These results indicate that the percentage of CD4(+) Foxp3(+) Treg cells are increased in H. pylori-infected mice, suggesting a role of Treg cells in H. pylori-induced pathologies, even at the early stages of chronic gastritis and gastric tumorigenesis.

Evaluation of p-phenylenediamine, o-phenylphenol sodium salt, and 2,4-diaminotoluene in the rat comet assay as part of the Japanese Center for the Validation of Alternative Methods (JaCVAM)-initiated international validation study of in vivo rat alkaline comet assay

As part of the Japanese Center for the Validation of Alternative Methods (JaCVAM)-initiated international validation study of in vivo rat alkaline comet assay (comet assay), p-phenylenediamine dihydrochloride (PPD), o-phenylphenol sodium salt (OPP), and 2,4-diaminotoluene (2,4-DAT), were analyzed in this laboratory as coded test chemicals. Male Sprague–Dawley rats (7–9 weeks of age) were given three oral doses of the test compounds, 24 and 21h apart and liver and stomach were sampled 3h after the final dose administration. Under the conditions of the test, no increases in DNA damage were observed in liver and stomach with PPD and OPP up to 100 and 1000mg/kg/day, respectively. 2,4-DAT, a known genotoxic carcinogen, induced a weak but reproducible, dose-related and statistically significant increase in DNA damage in liver cells while no increases were observed in stomach cells.

195 Rifaximin for the Treatment of Functional Dyspepsia: A Double-Blinded Randomized Placebo-Controlled Trial

and found that parietal cells in the infected stomach expressed IFNα/β protein in response to Helicobacter infection and that gastric IFNβ mRNA peaked 2 months after the infection while IFNα and IRF7 expression peaked at 2 to 4 months, followed by the peak in Slfn4 mRNA. Ex vivo IFNα11 (800U/ml) treatment of cultured peritoneal myeloid cells induced a phenotypic shift from Gr1-/Slfn4to Gr1+/Slfn4+ cells correlating with a robust increase in Slfn4 expression in vivo. Conclusion: Hedgehog signaling synergizes with Helicobacter infection to induce acquisition of Slfn4+ MDSCs in the stomach.

194 Eosinophils and Mast Cells in Functional Dyspepsia: Ultrastructural Evaluation of Activation

and found that parietal cells in the infected stomach expressed IFNα/β protein in response to Helicobacter infection and that gastric IFNβ mRNA peaked 2 months after the infection while IFNα and IRF7 expression peaked at 2 to 4 months, followed by the peak in Slfn4 mRNA. Ex vivo IFNα11 (800U/ml) treatment of cultured peritoneal myeloid cells induced a phenotypic shift from Gr1-/Slfn4to Gr1+/Slfn4+ cells correlating with a robust increase in Slfn4 expression in vivo. Conclusion: Hedgehog signaling synergizes with Helicobacter infection to induce acquisition of Slfn4+ MDSCs in the stomach.

Stimulation of Cystathionine‐γ‐lyase (CSE) Activity and Generation of Hydrogen Sulfide in Gastrointestinal Smooth Muscle via NO/cGMP/PKG pathway

Hydrogen sulfide (H2S) plays an important role in the regulation of gastrointestinal motility and secretion. H2S is synthesized mainly from L‐cysteine via cystathionine‐γ‐lyase (CSE) and cystathionine‐β‐synthase (CBS). We showed that smooth muscle cells of stomach and colon of rabbit, mouse and human express CSE, but not CBS. Activation of CSE causes inhibition of Rho kinase activity and muscle contraction and the inhibition was blocked by CSE siRNA and by a selective CSE inhibitor, propargylglycine (PPG).AimTo identify the mechanism of activation of CSE in gastrointestinal smooth muscle cells.MethodsActivation of CSE was analyzed by the measurement of H2S in response to L‐cysteine and NO donor sodium nitroprusside (SNP) in gastric and colonic muscle of rabbit and human. Filter paper disks dipped in zinc acetate were exposed to H2S trapped in N2‐perfused glass vials and the amount of reduced zinc acetate in the presence of H2S was measured spectrophotometrically.ResultsThe basal levels of H2S in gastric and colon muscle from rabbit and human were ~20‐25 nM/mg. L‐Cysteine (10 mM) increased H2S generation up to 50 nM/mg; the effect of L‐cysteine was inhibited by PPG. L‐cysteine‐induced H2S generation was augmented in the presence of SNP and cGMP analog, 8‐bromo cGMP to 125 µM/g and 120 nM/mg, respectively. Augmentation of CSE activity in response to SNP and 8‐br‐cGMP was blocked by the cGMP‐dependent protein kinase (PKG) inhibitor, Rp‐cGMPS, suggesting phosphorylation‐dependent activation.ConclusionWe have identified a mechanism of stimulation of CSE activity in gastric and colonic muscle involving cGMP‐dependent activation of PKG.

MiR-30-HNF4γ and miR-194-NR2F2 regulatory networks contribute to the upregulation of metaplasia markers in the stomach

ObjectiveIntestinal metaplasia and spasmolytic polypeptide-expressing metaplasia (SPEM) are considered neoplastic precursors of gastric adenocarcinoma and are both marked by gene expression alterations in comparison to normal stomach. Since miRNAs are...

Combination comet/micronucleus assay validation performed by BioReliance under the JaCVAM initiative

In the international validation study of the in vivo rat alkaline comet assay (comet assay), the Japanese Center for the Validation of Alternative Methods (JaCVAM) provided three coded chemicals to BioReliance, 1,3-dichloropropene, ethionamide and busulfan, to be tested in a combined in vivo comet/micronucleus assay.Induction of DNA damage (comet) in liver, stomach and jejunum (1,3-dichloropropene only) cells, and induction of MNPCEs in bone marrow, were examined in male Sprague–Dawley (Hsd:SD) rats following oral administration of the test chemical for three consecutive days. A dose range finding (DRF) test was performed with each chemical to determine the maximum tolerated dose (MTD). Based on the results of the DRF test; 1,3-dichloropropene was tested at 50, 100 and 200mg/kg/day; ethionamide was tested at 125, 250 and 500mg/kg/day, and busulfan was tested at 10, 20 and 40mg/kg/day.The results indicated that 1,3-dichloropropene induced DNA damage only in liver cells at all three test article doses, while no effects were observed in the stomach and jejunum cells. Additionally, it did not increase MNPCEs in the bone marrow. 1,3-Dichloropropene was concluded to be negative in the MN assay but positive in the comet assay. Ethionamide did not induce DNA damage in liver. However, in stomach, statistically significant decreases (although still within historical range) in % tail DNA at all test article doses compared to the vehicle control were observed. There was no increase in MNPCEs in the bone marrow. Thus, ethionamide was concluded to be negative in the comet/MN combined assay. Busulfan did not induce DNA damage in any of the organs tested (liver and stomach) but it did induce a significant increase in MNPCEs in the bone marrow. Busulfan was concluded to be negative in the comet assay but positive in the MN assay.

Comet assay evaluation of six chemicals of known genotoxic potential in rats

As a part of an international validation of the in vivo rat alkaline comet assay (comet assay) initiated by the Japanese Center for the Validation of Alternative Methods (JaCVAM) we examined six chemicals for potential to induce DNA damage: 2-acetylaminofluorene (2-AAF), N-nitrosodimethylamine (DMN), o-anisidine, 1,2-dimethylhydrazine dihydrochloride (1,2-DMH), sodium chloride, and sodium arsenite. DNA damage was evaluated in the liver and stomach of 7- to 9-week-old male Sprague Dawley rats. Of the five genotoxic carcinogens tested in our laboratory, DMN and 1,2-DMH were positive in the liver and negative in the stomach, 2-AAF and o-anisidine produced an equivocal result in liver and negative results in stomach, and sodium arsenite was negative in both liver and stomach. 1,2-DMH and DMN induced dose-related increases in hedgehogs in the same tissue (liver) that exhibited increased DNA migration. However, no cytotoxicity was indicated by the neutral diffusion assay (assessment of highly fragmented DNA) or histopathology in response to treatment with any of the tested chemicals. Therefore, the increased DNA damage resulting from exposure to DMN and 1,2-DMH was considered to represent a genotoxic response. Sodium chloride, a non-genotoxic non-carcinogen, was negative in both tissues as would be predicted. Although only two (1,2-DMH and DMN) out of five genotoxic carcinogens produced clearly positive results in the comet assay, the results obtained for o-anisidine and sodium arsenite in liver and stomach cells are consistent with the known mode of genotoxicity and tissue specificity exhibited by these carcinogens. In contrast, given the known genotoxic mode-of-action and target organ carcinogenicity of 2-AAF, it is unclear why this chemical failed to convincingly increase DNA migration in the liver. Thus, the results of the comet assay validation studies conducted in our laboratory were considered appropriate for five out of the six test chemicals.