Shorter Survival Interval Research Articles

Immunohistochemical detection of Fibronectin, P-Selectin, FVIII, HSP-70 and MRP-8 in the skin of ligature marks of suicidal hangings

The determination of vitality of skin injuries is one of the most central research areas in forensic pathology for it is often necessary to discern antemortem from postmortem damage. Typical is the case of a hanging, which should be distinguished from the postmortem suspension of a body. In this study, 15 human skin samples from ligature marks (study group) of victims of suicidal hanging and 15 uninjured skin samples (negative control group) were analyzed. In addition, 15 skin samples from ecchymoses in homicide victims with short survival intervals were investigated as a positive control group. Sections were processed for immunohistochemistry in order to detect the expression of Fibronectin, P-Selectin, FVIII, HSP-70, and MRP8. Immunohistochemical reactions were classified semiquantitatively (mild - score 1, moderate - score 2, and intense - score 3). In the ligature marks, Fibronectin was significantly less expressed compared to ecchymoses. The expression was similar to hanging marks and uninjured skin. In both ligature marks and ecchymoses, the expression of P-Selectin was significantly increased compared to uninjured skin. In both ligature marks and ecchymoses, expression of HSP-70 was significantly decreased in the epidermis compared to uninjured skin. In both ligature marks and ecchymoses, the expression of FVIII and MRP8 was significantly increased in the dermis and hypodermis compared to uninjured skin. The results of this study show that the immunohistochemical study of early inflammatory and coagulation factors could help determine the vitality of ligature marks. The combined analysis of P-Selectin, FVIII, HSP-70, and MRP-8 can be considered for this purpose.

Genomic Analysis Revealed Mutational Traits Associated with Clinical Outcomes in Osteosarcoma.

ObjectiveThe limited understanding of correlation between genomic features and biological behaviors has impeded the therapeutic breakthrough in osteosarcoma (OS). This study aimed to reveal the correlation of mutational and evolutionary traits with clinical outcomes.MethodsWe applied a case-based targeted and whole exome sequencing of eleven matched primary, recurrent and metastatic samples from three OS patients characterized by different clinical behaviors in local recurrence or systematic progression pattern.ResultsExtensive OS-associated driver genes were detected including TP53, RB1, NF1, PTEN, SPEN, CDKN2A. Oncogenic signaling pathways including cell cycle, TP53, MYC, Notch, WNT, RTK-RAS and PI3K were determined. MYC amplification was observed in the patient with shortest disease-free interval. Linear, branched or mixed evolutionary models were constructed in the three OS cases. A branched evolution with limited root mutation was detected in patient with shorter survival interval. ADAM17 mutation and HEY1 amplification were identified in OS happening dedifferentiation. Signatures 21 associated with microsatellite instability (MSI) was identified in OS patient with extra-pulmonary metastases.ConclusionOS was characterized by complex genomic alterations. MYC aberration, limited root mutations, and a branched evolutionary model were observed in OS patient with relatively aggressive course. Extra-pulmonary metastases of OS might attribute to distinct mutational process pertaining to MSI. Further research in a larger number of people is needed to confirm these findings.

Transcriptomic analysis reveals a WNT signaling pathway-based gene signature prognostic for non-small cell carcinoma.

The value of combining multiple candidate genes into a panel to improve biomarker performance is increasingly emphasized. Genes associated with WNT signaling are widely-reported to provide prognostic signatures in non-small cell carcinoma (NSCLC). Screening of genes involved in this signaling pathway facilitated selection of an optimal candidate biomarker gene combination and development of an NSCLC prognostic model based on expression of these genes. Risk scores derived from the model performed well in predicting survival; in the training dataset, samples achieving a high risk score exhibit a shorter survival interval (median survival time 34.8 months, 95% CI 31.1-41.0) than did samples achieving a low risk score (median survival time 72.0 months, 95% CI 59.3-87.5, p=2e-11), and exhibited higher oncogene and lower tumor suppressor gene expression. Receiver-operator characteristic curves based on three-year survival demonstrate that the model outperformed clinical prognostic indicators. In addition, the model was validated in four independent cohorts, demonstrating robust NSCLC prognostic value. Correlation analyses reveal that the model offers efficacy independent of other clinical indicators. Gene Set Enrichment Analysis (GSEA) reveals that the model reflects variable tissue functional states relevant to NSCLC biology. In summary, the signature model shows potential as a valuable and robust NSCLC prognostic indicator.

Short and long term effects of NMDA‐induced retinal excitotoxicity on melanopsin and non‐melanopsin containing retinal ganglion cells

AbstractPurposeTo study short and long term responses of the population of RGCs expressing Brn3a (Brn3a+RGCs) and the population of intrinsically photosensitive RGCs expressing melanopsin (m+RGCs) to excitotoxicity induced by intravitreal injection of N‐methyl‐D‐Aspartate (NMDA).MethodsIn adult albino Sprague Dawley rats, the left eye received an intraocular injection of 100nM NMDA and the retinas were analyzed 3 (n=9), 7 (n=6), 14 (n=10) days (d) or 15 (n=23) months (m) later. Spectral Domain Optical Coherence Tomography (SD‐OCT; Spectralis, Heidelberg) was used to image and analyze retinal thickness longitudinally in vivo at short (3 m; n=18) and long (15 m; n=18) survival intervals. Ex vivo, retinal whole mounts were immunodetected with antibodies against Brn3a and melanopsin and the numbers of surviving Brn3a+RGCs and of m+RGCs were automatically counted and represented using isodensity or neighbour maps.ResultsIntraocular injection of 100nM NMDA causes a massive loss of Brn3a+RGCs; at 3 and 14 days Brn3a+RGCs numbers diminished to 50% and 25%respectively, but there was no further loss up to 15 months. However, m+RGCs showed 3 d after injection a transient downregulation of melanopsin that recovered shortly and by 14 d and at the end of the study (15 m) the numbers of m+RGCs were comparable to their contralateral fellow eyes. SD‐OCT examination showed important reductions of the total and inner retinal thicknesses at 3 m that progressed further up to 15 m.ConclusionThe population of Brn3a+RGCs is quite sensitive to NMDA‐induced excitotoxicity that causes rapidly the loss of approximately 75% of these neurons. In contrast, m+RGCs appear fully resistant to NMDA‐induced excitotoxicity and show only a downregulation of melanopsin expression during the first two weeks. SD‐OCT can be used to assess retinal toxicity revealed by important reductions in total and inner retinal thickness at 3 m progressing up to 15 m.

YY1 expression predicts favourable outcome in follicular lymphoma

AimsFollicular lymphoma is the second most common type of non-Hodgkin's lymphoma worldwide. The majority of patients diagnosed as having follicular lymphoma have an indolent form of the disease, but a...

Heat shock protein 72 (Hsp72) improves long term recovery after focal cerebral ischemia in mice

Many brain protective strategies have been tested over short survival intervals, but few have been examined for long term benefit. The inducible member of the Heat shock protein 70 (Hsp70) family, Heat shock protein 72 (Hsp72), has been widely found to reduce ischemic injury. Here we assessed outcome in Hsp72 transgenic overexpressing mice and wild type littermates for one month following transient focal ischemia. Hsp72 reduced infarct area lost and improved behavioral outcome on rotarod and foot fault at one month. Thus protection by Hsp72 overexpression is long lasting, and includes improved recovery of motor function over one month.

Clinical quantitation of immune signature in follicular lymphoma by RT-PCR–based gene expression profiling

AbstractMicroarray gene expression profiling studies have demonstrated immune response gene signatures that appear predictive of outcome in follicular lymphoma (FL). However, measurement of these marker genes in routine practice remains difficult. We have therefore investigated the immune response in FL using real-time polymerase chain reaction (PCR) to measure expression levels of 35 candidate Indicator genes, selected from microarray studies, to polyA cDNAs prepared from 60 archived human frozen lymph nodes, in parallel with immunohistochemical analysis for CD3, CD4, CD7, CD8, CD10, CD20, CD21, and CD68. High levels of CCR1, a marker of monocyte activation, were associated with a shorter survival interval, and high levels of CD3 with better survival, while immunohistochemistry demonstrated association of high numbers of CD68+ macrophages with a shorter survival interval and of high numbers of CD7+ T cells with a longer survival interval. The results confirm the role of the host immune response in outcome in FL and identify CCR1 as a prognostic indicator and marker of an immune switch between macrophages and a T cell–dominant response. They demonstrate the utility of polyA DNA and real-time PCR for measurement of gene signatures and the applicability of using this type of “molecular block” in clinical practice.

Clinical Measurement of Prognostic Immune Signature in Follicular Lymphoma by RT-PCR Based Gene Expression Profiling and Immunohistochemistry Demonstrates Favourable T-Cell and Unfavorable Macrophage Infiltration.

Gene expression profiling studies have demonstrated immune response gene signatures predictive of outcome in follicular lymphoma (FL) and there is a need for validation of these signatures and for their translation to clinical use. However, measurement of these genes in routine practice remains difficult and to date there have been very few studies validating the hypothesis. We have previously demonstrated the utility of real-time PCR measurement of gene expression levels in globally amplified polyA cDNA as a clinically practical method for translation of gene signatures to clinical use. In this project we extended the method to analysis of immune response signatures in FL. We used real-time PCR to measure expression levels (normalised to the mean of 4 housekeeping genes) of 36 candidate Indicator genes, selected from microarray studies, in polyA cDNAs prepared using polyA PCR (method detailed in Sakhinia et al 2007) from 58 archived human frozen lymph nodes, together with immunohistochemistry for CD3, CD4, CD7, CD8, CD10, CD20, CD21 and CD68 in parallel formalin fixed paraffin embedded tissue samples to measure immune response in FL. Immunohistochemical positivity was measured by a semi-automated image analysis method using spectral unmixing to identify areas of immunopositivity. Kaplan-Mier survival analysis was performed against the normalised real-time PCR expression levels of each of the genes and against the percentage immunohistochemical postivity for CD3, CD4, CD7, CD8, CD10, CD20, CD21; for CD68 survival analysis was performed for cases with either 15 or less or more than 15 CD68 positive cells per high power field (hpf). High levels of CCR1, a marker of monocyte actication, were associated with a shorter survival interval (p<0.02) (figure 1a), whilst immunohistochemistry demonstrated association of high numbers of CD7 positive T-cells with longer survival interval (figure 1b) (p<0.032) and of high numbers of CD68 positive macrophages with a shorter survival interval (figure 1c) (p<0.02). The results confirm the role of the host immune response in outcome in FL and identify CCR1 as a prognostic indicator and marker of immune switch between macrophage and T-cell dominant response. The methods used are clinically applicable, whilst the clinical utility of polyA DNA and real-time PCR for measurement of gene signatures and the strength of this approach as a “molecular block” are confirmed. [Display omitted]

Clinical quantitation of diagnostic and predictive gene expression levels in follicular and diffuse large B-cell lymphoma by RT-PCR gene expression profiling

AbstractRecent microarray gene expression profiling studies have identified gene signatures predictive of outcome, so-called “indicator” genes, for diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL). However, measurement of these genes in routine practice remains difficult. We applied real-time polymerase chain reaction (PCR) to polyA cDNAs prepared from 106 archived human frozen lymph nodes (63 of FL, 25 of DLBCL, 10 reactive lymph nodes, and cases with paired samples of FL [4] and subsequent DLBCL [4]). Reverse transcription and polyA reverse transcriptase (RT)–PCR was performed, and resultant cDNA was probed by real-time PCR for 36 candidate indicator genes, selected from microarray studies. Nine genes showed statistically significant different expression between FL and DLBCL, including cyclin B, COL3A1, NPM3, H731, PRKCB1, OVGL, ZFPC150, HLA-DQ-a, and XPB. Of these, cyclin B, NPM3, and COL3A1 were higher in DLBCL. Six genes showed statistically significant higher expression in the neoplastic nodes compared with reactive nodes, namely PRKCB1, BCL-6, EAR2, ZFX, cyclin B, YY1. High levels of YY.1 were associated with a shorter survival interval in both FL and DLBCL. The method is simple, sensitive, and robust, facilitating routine use and may be used as a platform for clinical measurement of prognostic gene signatures.

YY1 Expression Predicts Survival in Follicular and Diffuse Large B-Cell Lymphoma.

Recent gene expression profiling has identified gene signatures predictive of outcome, Indicator genes, for diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL). However, measurement of Indicator genes in routine practice remains difficult. We have demonstrated utility of real-time PCR measurement of Indicator genes in globally amplified polyA cDNA as a practical method for their clinical analysis (Sakhinia et al 2005). PolyA PCR enables global mRNA amplification from picogram amounts of RNA and the polyA cDNA pool generated is indefinitely renewable, representing a “molecular block”. Real-time PCR measurement of the expression levels of specific Indicator genes then allows gene signatures to be detected in the polyA cDNA. In this project we applied real-time PCR for 36 Indicator genes, to polyA cDNAs prepared from 122 archived human frozen lymph nodes; specifically 63 cases of FL, 25 of DLBCL and 10 reactive lymph nodes were analysed; the remaining 24 cases were FL with either evidence of focal transformation, of subsequent transformation or (4 cases) paired frozen samples of FL and subsequent DLBCL. PolyA RT-PCR was performed on extracted RNA and resultant cDNA probed for 36 candidate Indicator genes (selected from Husson et al 2002, Shipp et al 2002 and Rosenwald et al 2003), by real-time PCR with quantification against human DNA, and normalisation to the mean of four housekeeping genes. Statistical analysis was performed using the Mann Whitney test and Kruskal-wallis with a p≤ 0.05 for statistical significance; all results detailed below are significant to at least p≤ 0.05. Ten genes showed statistically significant different expression between FL and DLBCL, including Cyclin B, COL3A1, NPM3, H731, PKC.B1, OVGL, ZFPC150, HLA-DQ-a, and XPB. Of these, cyclin b, a cell cycle gene, NPM3, nucleolar phosphoprotein, and COL3A1were higher in DLBCL. Six genes showed statistically significant higher expression in the neoplastic nodes compared to reactive nodes, namely PKCB-1, BCL-6, EAR2, ZFX, Cyclin B, YY.1. Comparison of gene expression levels in cases of FL that either did not or did subsequently transform to DLBCL demonstrated significant upregulation of ACTA, HLA-DQ-a in the latter cases of FL. High levels of YY.1 were associated with a shorter survival interval in both FL and DLBCL by Kaplan-meir survival analysis. This is of particular interest given the reported role of YY.1 in producing resistance to Rituximab due to downregulation of FAS -induced apoptosis (Vega et al, 2005). This was reported for a cell culture study and has not been shown in clinical studies, though the result in this project suggests a possible detrimental role for YY.1 clinically and that it may act as a predictor of Rituximab response. These results :demonstrate the possibility of using polyA PCR for global amplification of clinical samples and real-time PCR to measure diagnostically informative gene expression profiles in the resultant polyA cDNA “molecular block” andidentify novel prognostic markers for FL and DLBCL.The method is simple, sensitive and robust, and amy be used as a platform for clinical measuremen of prognostic gene signatures. Whilst lymphoma represents a relatively small group of cancer patients the generic nature of microarray gene profiles for cancer subtypes will facilitate simple extension of the method to other tumour types.

Noncoding miRNAs as novel prognostic factor for 5-fluorouracil adjuvant therapy in colorectal cancer

3609 Background: The roles of non-coding miRNAs in tumorgenesis are just emerging. miRNAs regulate gene expression at post-transcriptional level by influencing translation efficiency of their target mRNAs. Previous studies from our laboratory have identified a number of miRNAs that were dis-regulated in colon cancer cell lines related to the loss of p53 tumor suppressor gene. In this study, the in vivo significance of some of these miRNAs was further evaluated using clinical samples. Ten miRNAs (hsa-let-7b, hsa-let-7g, hsa-miR-15b, hsa-miR-181b, hsa-miR-191, hsa-miR-200c, hsa-miR-26a, hsa-miR-27a, hsa-miR-30a-5p and hsa-miR-30c) were evaluated for their potential prognostic value in colorectal cancer patients receiving 5-fluorouracil (5-FU) based therapy. Methods: Forty eight snap frozen clinical colorectal samples (24 normal and 24 paired colorectal cancer patient samples) were selected with detailed clinical follow-up information. RNAs were isolated from these samples using TRIzol reagent. After cDNA synthesis with miRNA specific primers, the expression levels of 10 miRNAs were quantified via qRT-PCR analysis. The statistical significance of these markers for disease prognosis was evaluated using two tailed paired T-test. Kaplan-Meier survival curve was generated and followed by Logrank test. Result: Among the ten miRNAs, hsa-miR-15b (p=0.02), hsa-miR-181b (p=0.01), hsa-miR-191 (p=0.03) and hsa-miR-200c (p=0.005) were significantly over-expressed in tumors compared to normal colorectal samples. Kaplan-Meier survival analysis indicates that hsa-miR-200c was a significant prognostic marker for predicting patient’s survival (p=0.01). The patients (n=15) with higher hsa-miR-200c expression had shorter survival interval (median survival = 26 months) compared to patients (n=9) with lower expression (median survival = 38 months). Conclusions: Some of these miRNAs may function as oncogenes due to their over-expression in tumors. Hsa-miR-200c may be a potential novel prognostic factor for 5-FU based chemotherapy in colorectal cancer. No significant financial relationships to disclose.

Serotonin Transporter Polymorphisms in Familial and Idiopathic Pulmonary Arterial Hypertension

Serotonin is a pulmonary vasoconstrictor and smooth muscle cell mitogen. The serotonin transporter (SERT) is abundant in pulmonary vascular smooth muscle. Compared with the short (S) allele, the long (L) SERT promoter allele is associated with increased SERT transcription and more severe pulmonary hypertension in a cohort of patients with chronic obstructive pulmonary disease, and was more prevalent in a cohort with idiopathic pulmonary arterial hypertension (IPAH), compared with control subjects. We hypothesized that the SERT L allele would associate with an earlier age at diagnosis and/or shorter survival interval in pulmonary arterial hypertension (PAH) than the S allele. SERT promoters from 166 familial PAH (FPAH), 83 IPAH, and 125 control subjects were sequenced. One hundred twenty-seven of the patients with FPAH had a known mutation in bone morphogenetic protein receptor 2 (BMPR2). The mean age at diagnosis was 35.8 yr in patients with FPAH and 41.1 yr in patients with IPAH (p = 0.02). There were no significant differences in distribution of the LL, LS, or SS genotypes in IPAH, FPAH, or unaffected BMPR2 mutation carriers. In FPAH, the LL genotype was associated with an earlier age at diagnosis (p < 0.02). In patients with IPAH, these SERT genotypes do not correlate with age at diagnosis or survival interval. In patients with FPAH, the LL genotype correlates with an earlier age at diagnosis than SL or SS, although survival among the groups was similar. The correlation of the SERT promoter polymorphism with age at diagnosis in FPAH suggests a possible relationship between the SERT and BMPR2.

Disposition of metronidazole in hens (Gallus gallus) and quails (Coturnix coturnix japonica): pharmacokinetics and whole-body autoradiography.

Hens were given single intravenous or oral doses (30 mg/kg body weight) of metronidazole and the plasma concentrations of the drug were determined by high-performance liquid chromatography (HPLC) at intervals from 10 min to 24 h after drug administration. Pharmacokinetic variables were calculated by the Lagrange algorithm technique. The elimination half-life (t1/2 beta) after the intravenous injection was 4.2 +/- 0.5 h, the volume of distribution (Vd(ss) 1.1 +/- 0.2 L/kg and the total body clearance (ClB) 131.2 +/- 20 mL/h.kg. Oral bioavailability of the metronidazole was 78 +/- 16%. The plasma maximum concentration (Cmax) 31.9 +/- 2.3 micrograms/mL was reached 2 h after the oral administration and the oral elimination half-life (t1/2 beta) was 4.7 +/- 0.2 h. The binding of metronidazole to proteins in hen plasma was very low (less than 3%). Whole body autoradiography of [3H] metronidazole in hens and quails showed an even distribution of labelled material in various tissues at short survival intervals (1-4 h) after oral or intravenous administration. A high labelling was seen in the contents of the small and large intestines. In the laying quails a labelling was also seen in the albumen and in a ring in the periphery of the yolk at long survival intervals. Our results show that a concentration twofold above the MIC is maintained in the plasma of hens for at least 12 h at an oral dose of 30 mg/kg metronidazole.

Preexposure and Subsequent Resistance to Lead in Yearling Brook Trout Salvelinus fontinalis

Potential for acclimation of brook trout, Salvelinus fontinalis, at sublethal levels of dissolved lead was measured. Samples of yearlings (19.2 ± 6.1 g) were preexposed continuously to environmentally realistic sublethal levels (10, 24, and 37 μg Pb/L) for 10, 20, 30, and 40 d and subsequently exposed to a lethal level (2 mg Pb/L), during which the incidence of mortality was recorded. Survival intervals of fish preexposed to 10 μg Pb/L were not significantly different from those preexposed to tap water (&lt;2 μg Pb/L) in each preexposure interval. Fish preexposed to 24 μg Pb/L for 10 and 20 d were less resistant but returned to normal resistance after 30 and 40 d of preexposure. Fish preexposed to 37 μg Pb/L had significantly shorter survival intervals than those preexposed to tap water in each preexposure interval. The maximum acceptable toxicant concentration (MATC) of lead for brook trout is set between 24 and 37 μg Pb/L.

Distribution of 64Cu in foetal and adult tissues in mice: influence of sodium diethyldithiocarbamate treatment.

64Cu (as 64CuCl2) was given intravenously to male C57BL mice and to pregnant C57BL mice at various stages of gestation. The disposition of the 64Cu in the adult animals and in the foetuses was studied by autoradiography and gamma spectrometry. The effects of treatments with diethyldithiocarbamate (DEDTC) on the disposition of the 64Cu in the animals were also examined. In addition, the ability of Cu to affect chondrogenesis was studied in an embryonic limb bud culture system. The results showed a strong uptake of 64Cu in the liver of the adult animals at all intervals (5 min.-24 hrs). At short survival intervals, there was also an uptake in the kidney cortex, the gastrointestinal mucosa, the adrenal, the pancreas, and the erythrocytes. Exposure to Cu may cause liver and kidney injuries, which may be related to the strong accumulation in these organs. 64Cu passed the placenta to the foetuses at all stages of gestation, although this occurred at a relatively slow rate. Within the foetuses the highest concentrations were found in the liver. Cu was observed to be toxic in the chick limb bud mesenchymal spot culture system although at relatively high concentrations. Foetal malformations and embryotoxicity may therefore be interpreted as a result of direct action of Cu on embryonic structures, although placental and/or maternal influence cannot be excluded. Pre- or posttreatment of the animals with DEDTC, which is a chelating agent, caused a very marked increase in the concentration of 64Cu in most tissues of the adult animals and also an increased foetal uptake of the metal.(ABSTRACT TRUNCATED AT 250 WORDS)

Accumulation in murine amniotic fluid of halothane and its metabolites.

The distribution of radioactivity in pregnant mice was registered at 0, 4, and 24 hrs after a 10 min. period of inhalation of 14C-halothane. Autoradiographic methods were used to allow to distinguish between the distribution of volatile (non-metabolize) halothane, water-soluble metabolites, and firmly tissue-bound metabolites. While volatile radioactivity was seen predominantly at short survival intervals, e.g. in body fat, blood, brain and liver, metabolites accumulated with time. Peak values occurred at 4 hrs in most organs (measured with liquid scintillation as well). The most remarkable findings were the high concentrations of radioactivity in amniotic fluid (and the ocular fluids of adults) with peak values at 4 hrs and rather high concentrations still prevailing at 24 hrs after inhalation. It is assumed that this activity represents only partly volaile halothane and mostly non-volatile metabolites. High activity of metabolites was seen in the neuroepithelium of the embryo in early gestation. Firmly tissue-bound metabolites, still remaining after washing the tissues with trichloroacetic acid and organic solvents, were found in the nasal mucosa, trachea and bronchial tree and in (presumably centrilobular) zones of the liver of adults after inhalation and 5-day old mice after intraperitoneal injection, indicating the formation of reactive metabolites in these organs. Firmly tissue-bound activity was not observed in the corresponding foetal organs.

The Tissue‐Disposition of Gentamicin in Rats

Abstract: The tissue disposition of 14C‐gentamicin was studied in young rats after a single‐dose administration by autoradiographic techniques. The autoradiograms showed a high accumulation of the substance in non‐parenchymateous tissues such as cartilages and connective tissues and a strong uptake in the kidney cortex. The in vivo bound substance was displaced from the cartilage at incubations in cation‐containing solutions, indicating an ionic binding of the polycationic gentamicin to negatively charged groups in the cartilage (probably carboxyl and sulphate groups of chondroitin sulphate). In the inner ear a localization of the substance was found in the membraneous walls of the chochlea and, at short survival intervals, in the perilymph, whereas the radioactivity in the endolymph was low. Studies in vitro showed an ionic binding of gentamicin to cartilage and in addition to several other tissues, such as the lymphomyeloid system, the gastrointestinal mucosa, the exocrine pancreas, salivary glands, and most prominent, the pigmented tissues. A strong in vitro binding of gentamicin to pigment from beef eyes was also shown. The potential binding sites in the latter tissues are localized intracellularly and are therefore apparently unattainable for the charged hydrophilic gentamicin molecule in vivo.

Sites of metabolism of N-nitrosodiethylamine in mice

Low-temperature whole-body autoradiography and autoradiography with heated sections in C57B1 mice injected with N-[ 14C]nitrosodiethylamine showed a homogenously distributed volatile radioactivity in most tissues — indicating an ability of the non-metabolized substance to freely pass the biological membranes and distribute evenly in the intra- and extracellular tissue-water. A high level of non-volatile metabolites was found in several tissues: the nasal and tracheal mucosa, the mucosa of the bronchial tree, the salivary glands, the liver, the mucosa of the oesophagus and the tongue, and the lacrimal glands. Studies in vitro indicated that these tissues had a capacity to degrade N-[ 14C]nitrosodiethylamine ( 14CO 2-production and incorporation of radioactivity in the acid-insoluble material of the tissues were used as indices of the metabolism), whereas several other tissues, which did not accumulate metabolites at short survival intervals in vivo, were devoid of significant metabolic capacity. The relationship between metabolic ability and carcinogenic response of the tissues for N-nitrosodiethylamine is discussed on basis of the obtained results.

The distribution of [ 14C]dimethylnitrosamine in mice. Autoradiographic studies in mice with inhibited and noninhibited dimethylnitrosamine metabolism and a comparison with the distribution of [ 14C]formaldehyde

The distribution of [ 14C]dimethylnitrosamine was studied by wholebody autoradiography in mice. By applying low temperature autoradiography and autoradiography with dried tape sections in mice with inhibited (by pretreatment with pyrazole, ethanol, or nialamide) and noninhibited dimethylnitrosamine metabolism, a distinction was possible between the distribution of the volatile nonmetabolized dimethylnitrosamine and the nonvolatile metabolites. The results indicated a uniform distribution of the nonmetabolized dimethylnitrosamine in the body. At the short survival intervals, metabolites were trapped primarily in the liver and also to a low degree in the kidneys, probably reflecting the alkylating reactions and other reactions connected with the breakdown of dimethylnitrosamine. At the long survival intervals, metabolites also occurred in tissues with a rapid cell turnover and a high rate of protein synthesis. Since autoradiography of [ 14C]formaldehyde gave this distribution picture, incorporation of radioactivity from [ 14C]dimethylnitrosamine via [ 14C]formaldehyde in the one-carbon pool probably explains the labeling of these tissues. Pyrazole, ethanol, and nialamide strongly depressed the incorporation of radioactivity from [ 14C]dimethylnitrosamine into the acid-insoluble material of the liver. In addition, the pretreatments caused a strong inhibition of the exhalation of 14CO 2 from [ 14C]dimethylnitrosamine.