miR-21 and miR-155 are associated with mitotic activity and lesion depth of borderline melanocytic lesions.

BACKGROUND: Expression of microRNAs (miRs) has been shown to be altered in many solid tumours and is being explored in melanoma. The malignant potential of some melanocytic lesions is difficult to predict. We hypothesised that characterisation of miR expression in borderline melanocytic proliferations would lead to the identification of a molecular profile that could be used with known prognostic factors to differentiate lesions with high malignant potential. METHODS: The miR expression profile of melanocytic lesions (benign naevi, malignant melanoma and borderline melanocytic tumours) was evaluated by real-time PCR. RESULTS: PCR analysis revealed primary cutaneous melanomas had an 8.6-fold overexpression of miR-21 and a 7.5-fold overexpression of miR-155 compared with benign naevi (P<0.0001). In situ hybridisation confirmed these results. miR-21 and miR-155 were significantly overexpressed within borderline lesions (P=0.0011 and P=0.0048, respectively). When borderline lesions were categorised by mitotic activity and Breslow thickness, miR-21 was associated with mitotic activity and miR-155 was associated with thickness (P<0.025). Among 14 patients with borderline lesions who underwent sentinel lymph node biopsy (SLNB), positive SLNB was associated with increased miR-21 and miR-155 in the primary lesion compared with lesions with a negative SLNB. CONCLUSION: MicroRNA expression profiles can be used to characterise atypical melanocytic lesions.

Shiftwork and industrial injuries at a chemical plant in southeast Texas.

A historical retrospective study of 242 shiftworker (SW) and 224 non-SW (NSW) injuries at a chemical manufacturing plant in southeast Texas (U.S.A.) was performed. Data were collected on injuries that occurred between 1 January 1982 and 31 December 1985. The SW schedule comprised an 8-hr, 7-day backward rotation program while the dayworker schedule consisted of a typical U.S.A. 40-hr work week. Injury records were matched against payroll/attendance records to substantiate the probability of isolating possible effects of the shiftwork rotation schedule on the rates, distribution and severity of injuries. Injuries in the SW sample were compared to those in the non-SW sample. The work responsibilities of dayworkers were not exactly the same as those of shiftworkers. However, the job responsibilities for males and females within the dayworker and shiftworker groups were equivalent. Overall injury incidence rates were not significantly different between SWs and non-SWs, although female SWs had significantly higher incidence rates than did male SWs and both male and female dayworkers. The occurrence of injuries, however, was not equally distributed during the day shift and the night shift, but was equally distributed during the evening shift and for non-SWs, suggesting that certain, yet to be determined, factors were affecting the distribution of injuries during the day and night shift. However, the average number of injuries was two-three times higher during the first four days of the day and night shift, yet were not elevated during the evening shift.

The influence of inhaled formaldehyde on rat lung cytochrome P450.

The effect of formaldehyde (HCHO) inhalation on total cytochrome P450 in the lungs of Sprague-Dawley rats was assessed after single and repeated exposures to 0, 0.5, 3, and 15 ppm HCHO. Whole-body exposures were conducted in dynamic, monitored exposure systems for 6 hr/day, 5 days/week, for periods of exposure of 1 day, 4 days, 12 weeks, or 24 weeks. Lung microsomal fractions were prepared and total protein and cytochrome P450 were measured 18 hr after the end of exposure at each time point. Two separate sets of exposure studies were conducted, thus duplicating all measurements for each dose group and at each time point. There were no detectable levels of total lung P450 in any of the rats that received a single 6-hr exposure to all three HCHO doses, while control lung P450 levels were similar to that found for 4-day and 12-week control rats. After 4 days of repeated exposures, however, there was a highly significant, reproducible, and dose-dependent increase in lung P450 levels relative to controls, with the 0.5, 3, and 15 ppm groups demonstrating 387, 1026, and 1123% of control values, respectively. Lung P450 levels remained elevated at all HCHO concentrations through 12 and 24 weeks of exposure, although the percentage difference between exposed and control rats continually dropped throughout the course of long-term repeated exposures. While HCHO-exposed rats did have decreased total body weight relative to controls, lung microsomal protein and lung weight of nearly all of the HCHO-exposed rats was not significantly different from the controls. The initial inactivation of lung P450 after a single HCHO exposure is apparently a transient phenomenon, with dose-dependent induction of the total P450 levels in the lung as the pattern of response to repeated exposures to inhaled HCHO.

The mutagenicity of urine fractions from patients administered antineoplastic therapy.

A concern among hospital personnel is their exposure to mutagenic drugs and in the incidental exposures that could occur in caring for the patients. In a recent published study the mutagenicity of urine from patients administered antineoplastic drugs was determined and techniques were developed to chemically inactivate the mutagenicity. A question still remained as to what components of the excreted urine were mutagenic. Urine samples from patients receiving mutagenic drugs were fractionated by high pressure liquid chromatography (HPLC) to then assay by the Ames test the collected and concentrated fractions to determine what were the mutagenic compounds in the urine. Urine samples from patients on single agent cancer treatment with cisplatin, cyclophosphamide, doxorubicin and mitomycin C were assayed. In general, all urine samples containing the cytotoxic agents studied were mutagenic because of the presence of the parent compound, except cyclophosphamide which requires activation and therefore an active metabolite was the major mutagenic constituent in the urine sample. This data indicates that the mutagenicity of urine from patients receiving these antineoplastic agents is the result of the parent compound or a single major metabolite.

Distribution of DNA and RNA content in the bone marrow and alveolar macrophages of rats after subchronic inhalation of formaldehyde.

The technique of flow cytometry was used to monitor the cell-cycle distribution of DNA and RNA in selected tissues of rats that were subchronically exposed to formaldehyde (HCHO) inhalation. Male Sprague-Dawley rats inhaled HCHO vapor concentrations of 0, 0.5, 3, or 15 ppm for 6 hr/day, 5 days/week, for up to 24 weeks. Simultaneous two-parameter measurements were made on a Phywe ICP 22 pulse cytophotometer by use of an acridine orange stain of the DNA and RNA of each cell sampled. No significant changes relative to the controls were determined in the percentage S and G2 + M phases of the DNA from sample tissues of the HCHO-exposed animals. Increases of 50 to 60% in the RNA content of G1 cells (RI) in the alveolar macrophages were seen after 1 week of exposure at all three HCHO doses. This effect was diminished after subchronic exposure. No HCHO-related effect was observed, though, in the RI of the rat bone marrow cells at any time point. However, the observed changes in RNA content were of rather limited magnitude relative to the cell-cycle perturbation induced by known cytotoxic agents in this and other flow cytometry studies. These modest and transient RI increases therefore probably did not reflect any significant effect on cell viability or cell proliferation in the affected tissues.

Induction of metallothionein synthesis in human peripheral blood leukocytes.

Metallothionein (MT), a low molecular weight, metal-binding protein, has recently been shown to protect murine mononuclear phagocytic cells from the cytotoxic effects of bacterial lipopolysaccharides (LPS), the endotoxic component of Enterobacteriaceae. MT appears to function intracellularly as an antioxidant since autolysis results from lipid peroxidation initiated by free radicals of O2. Since this activity is distinct from MT's capacity to specifically sequestrate heavy metals, we examined whether MT synthesis can be induced by direct membrane activation or through interaction with soluble leukocyte mediators. Normal human monocytes, polymorphonuclear neutrophils (PMN), and lymphocytes, isolated from heparinized whole blood, were incubated with and without LPS from Escherichia coli and Salmonella typhosa. MT in cell lysates was quantitated using a 203Hg-binding assay employing Sephadex G-10 "minicolumns." When incubated with monocytes, PMN, or lymphocytes, neither preparation of LPS (10-100 micrograms/ml) was capable of enhancing 203Hg-binding activity after 24 or 72 hr incubation. CdCl2 (2 micrograms/ml), however, increased binding activity in monocyte and lymphocyte cultures 4- and 15-fold, respectively. When monocytes and lymphocytes were cocultured with LPS, 203Hg-binding activity was not enhanced. Addition of human interleukin 1 (endogenous pyrogen) to these cultures had no significant effect. Leukocyte endogenous mediator (LEM), a product of LPS-activated PMN that possesses hypozincemic activity in vivo, did not induce MT synthesis. Collectively, these results demonstrate that leukocyte MT does not arise from direct LPS activation or from interaction with products secreted by LPS-activated cells. De novo synthesis of MT observed during endotoxemia and gram negative sepsis appears, therefore, to be induced by endogenously released corticosteroids.

Toxicologic interactions between ozone and bacterial endotoxin.

The effects of acute exposure of mice to bacterial lipopolysaccharide (LPS), the endotoxin of gram negative microorganisms, and ozone (O3) have been investigated. Intraperitoneal (ip) administration of 5 mg/kg LPS to CD-1 mice followed by exposure to 15 ppm O3 for 1.5 hr produced synergistic effects as measured by pulmonary edemagenesis and lethality assays. In contrast, ip administration of 0.1-1.6 mg/kg LPS to CD-1 mice over 5 consecutive days, a dose regimen resulting in LPS tolerance, protected against a lethal challenge of 20 ppm O3 for 3 hr. A statistically significant increase in catalase and glutathione peroxidase activity was measured in homogenates of lungs obtained from CD-1 mice receiving a tolerance-inducing regimen of LPS. These results demonstrate that two, distinct toxicologic interactions can occur between O3 and bacterial LPS. Synergism between these agents could explain, in part, the increased susceptibility of O3-exposed animals to respiratory infection with gram negative microorganisms. Protection resulting from LPS-induced increases in pulmonary antioxidant activity provides additional evidence that O3 and, possibly, LPS mediate their toxicity through oxidative mechanisms.

Stability and inactivation of mutagenic drugs and their metabolites in the urine of patients administered antineoplastic therapy.

Urine samples from patients administered mutagenic antineoplastic drugs are mutagenic in the Ames assay, and hence may pose a genotoxic hazard to hospital personnel or family members caring for the patient. The urine samples in the present study were tested for mutagenicity in several strains of Salmonella typhimurium that were uvr negative (TA98, TA100) or positive (TA102, UTH8413, UTH8414), and were analyzed for the presence of drugs and their metabolites using high-pressure liquid chromatography (HPLC). Urine samples from cancer patients were kept at room temperature and their mutagenicity as well as the chemical stability of the drugs was tested for a period of 14 days. It was observed that, in general, the urine remained mutagenic for the 14-day period while the parent compound degraded within the first seven days. An exception was cisplatin, which was chemically stable as platinum, but the urine decreased in mutagenicity with time. This decrease was probably the result of ligand exchange with the platinum. Inactivation methods were developed to reduce the genotoxic hazard posed by the mutagenic compounds in the urine. Cisplatin was inactivated by complexing with sodium diethyldithiocarbamate (DDTC). Oxidation of urine containing mitomycin C and doxorubicin (sodium thiosulfate must be added to urine containing doxorubicin) with 5.25% sodium hypochlorite solution (bleach) results in mutagenic inactivation. Urine containing cyclophosphamide and its metabolites was oxidized with alkaline potassium permaganate and the active degradation products trapped with sodium thiosulfate. Both chemical and mutagenic assays are necessary to determine the reduction of risk. Methods of inactivation of mutagenic urine developed in this study are both effective and practical for the reduction of exposure to genotoxic hazards.

Evidence for lipid peroxidation in endotoxin-poisoned mice.

Ethane has been identified and quantitated in air exhaled by mice following intraperitoneal injection of 20, 40, or 200 mg of Escherichia coli O111:B4 lipopolysaccharide (LPS) per kg. Significant increases in ethane concentration occurred within 1 to 5 h after LPS administration. In addition, increased concentrations of malondialdehyde were found in crude homogenates of livers obtained from mice 16 h after administration of 20 mg of LPS per kg. These results suggest that lipid peroxidation may be an important mechanism responsible for LPS toxicity.

Respiratory responses in the lower respiratory tract of Sprague-Dawley rats to formaldehyde inhalation.

This study was conducted to determine the characteristic response of Sprague-Dawley rats to formaldehyde (HCHO) challenges to the lower respiratory tract, and whether these response patterns are altered in rats that have received repeated exposures to HCHO. Male Sprague-Dawley rats were exposed to 0, 0.5, or 15 ppm HCHO for 6 hours/day, 5 days/week, for 8 or 16 weeks. Both naive rats and rats repeatedly exposed to HCHO were then administered 30 ppm HCHO test challenges by tracheal exposure, with the minute volume, respiratory rate, and tidal volume responses monitored. The pulmonary response of naive rats to HCHO tracheal challenge involved the correlation of minute volume and tidal volume depression, while respiratory rate was either unaffected or slightly increased. This was also the response pattern for rats that received 8 weeks of repeated exposure to HCHO. The only significant difference in respiratory response patterns between naive and pre-exposed animals existed in a slight increase in the respiratory rate compensatory response in the rats pre-exposed for 16 weeks to 15 ppm. There was substantial recovery of initially depressed respiratory parameters during the tracheal challenge in both naive and pre-exposed rats. The characteristic pulmonary response to HCHO in the lower respiratory tract demonstrated for Sprague-Dawley rats was thus similar to patterns of lower respiratory response to HCHO reported for other rodent species.

Synthetic pyrethroid insecticides: a dermatological evaluation.

Synthetic pyrethroids are lipophilic insecticides whose biological activity seems to be directly related to their chemical structure. In this investigation differences in cutaneous sensation were detected by human participants between synthetic pyrethroids with a cyano group in the (S)-configuration of the 3-phenoxybenzyl alcohol of their molecular structure (fenvalerate) and those that do not (permethrin). A strong relation was noted between insecticidal potency and degree of induced cutaneous sensation for the alpha-cyano and non-cyano pyrethroids, with a prominent difference between the two. No sensation was observed by any of the same participants on topical exposure to the inert ingredients of these agents. A linear correlation between concentration and degree of induced dysaethesia was observed for both pyrethroids. Regressing the cutaneous sensation on the common logarithm of concentration resulted in a regression equation of Y = 84.0 + 31.0X1 for fenvalerate and Y = 27.5 + 15.8X1 for permethrin. A highly efficacious therapeutic agent for pyrethroid exposure was noted to be dl-alpha tocopherol acetate. An impressive degree of inhibition of paraesthesia resulted from the topical application of vitamin E acetate, with a therapeutic index of almost 100%.

Primary irritant contact dermatitis from synthetic pyrethroid insecticide exposure.

Synthetic pyrethroids are widely used insecticides with numerous applications, varying from food protection to general pest control. Humans are capable of tolerating greater acute and chronic exposures to the pyrethroids than to many other insecticides. An abnormal cutaneous sensation (paresthesia) is known to occur after dermal contact with the pyrethroids. Recent field studies have indicated that a primary irritant contact dermatitis may also develop. This investigation evaluated dermal irritancy from cutaneous synthetic pyrethroid application to albino rabbits. Through repeated daily applications of either fenvalerate or permethrin, a slight erythema was noted visually which correlated with increased cutaneous blood flow measured by laser Doppler velocimetry. Histopathological changes were also documented, but no significant differences were detected in edema or thermal variation.

Effect of subchronic formaldehyde inhalation on minute volume and nasal deposition in Sprague-Dawley rats.

Since respiratory depression during formaldehyde (HCHO) inhalation is an important mechanism in reducing the dose received and potentially the toxicity in the nasal passages of exposed animals, this study was conducted to determine if changes in the pattern of minute volume response and nasal deposition occurred in nosepiece challenges to rats after long-term repeated exposures to HCHO. Male Sprague-Dawley rats were exposed to 0, 0.5, 3, or 15 ppm HCHO for 6 h/d, 5 d/wk, for 8 or 16 wk. The preexposed animals and age-specific controls were then submitted to a HCHO nosepiece challenge at the same concentration that was received in the subchronic exposure. Very high nasal deposition was demonstrated in all measurements. There was a diminished maximum minute volume depression in the 16-wk group relative to the 8-wk group. The difference in response was not statistically associated with the subchronic preexposure concentration. The substantial recovery of all initially depressed responses that occurred during the challenges probably diminished the impact of the decreased maximum responses on the resulting nasal deposition over the course of the long-term exposures.

Concentration-time course of niridazole and six metabolites in the serum of four Filipinos with Schistosoma japonicum infection.

Niridazole and six of its metabolites have been quantitated by high-pressure liquid chromatography in sera of four male Filipino patients with mild Schistosoma japonicum infections given single oral doses of niridazole (15 mg/kg) on two occasions 10 days apart. Of the five oxidative metabolites measured, 4-hydroxyniridazole and 4-ketoniridazole achieved the highest concentrations, reaching peak values of 0.9 +/- 0.3 microgram/ml of serum (mean +/- S.D., n = 4) and 0.7 +/- 0.1 microgram/ml of serum within 1 to 4 hr. 4-Ketoniridazole achieved peak serum levels 1 hr after the other oxidative metabolites in three of four patients and was the predominant metabolite in the serum of all patients 6 to 10 hr after dosing. By 24 hr, both 4-ketoniridazole and 4-hydroxyniridazole had largely disappeared from the serum. Niridazole and three other oxidative metabolites, 4,5-dihydroxyniridazole, 5-hydroxyniridazole and 4,5-dehydroniridazole, appeared within 1 hr in serum but failed to exceed 0.4 microgram/ml; none of these compounds were detected in the 24-hr serum samples. The pharmacokinetic pattern of niridazole and the oxidative metabolites showed marked interindividual variation but was quite reproducible in the same individual studied 10 days later. 1-Thiocarbamoyl-2-imidazolidinone was analyzed in serum samples by a different high-pressure liquid chromatographic procedure. This reductive metabolite attained maximal levels of 50 to 150 ng/ml of serum 6 to 12 hr after drug administration and remained at 40% or more of its peak concentration even after 24 hr.(ABSTRACT TRUNCATED AT 250 WORDS)

4-Keto niridazole: a major niridazole metabolite with central nervous system toxicity different than niridazole.

4-Keto niridazole, isolated by high-pressure liquid chromatography, was identified by high resolution electron impact mass spectral analysis as a major drug metabolite of niridazole in the serum or plasma of rats and mice treated orally or i.p. with niridazole. This metabolite has a pKa of 5.8 and is approximately 40% bound at physiologic pH to serum proteins of mice receiving therapeutic doses of niridazole. After i.p. injection of niridazole (160 mg/kg), peak serum levels of 4-keto niridazole (10.4 micrograms/ml) were reached within 6 hr in DBA/2J mice. The acute LD50 for 4-keto niridazole i.p. was 55 mg/kg in DBA/2J mice and 51 mg/kg in C57BL/6J mice; the comparable value for niridazole was 220 mg/kg in DBA/2J mice. Signs of acute 4-keto niridazole toxicity were different from those of niridazole toxicity and consisted of profound sedation and labored, irregular breathing terminating in respiratory arrest. Daily i.p. injection of 30 mg/kg of 4-keto niridazole for 5 days into DBA/2J mice resulted in no evidence of cumulative toxicity. The serum and brain concentrations of 4-keto niridazole after a 70-mg/kg i.p. LD90 dose of this compound were 93 micrograms/ml and 7.5 micrograms/g just before death. If an LD90 dose of niridazole (285 mg/kg) was injected into DBA/2J mice, the serum and brain concentrations of 4-keto niridazole just before death were 15 and 5%, respectively, of those found after an LD90 dose of 4-keto niridazole. Thus, 4-keto niridazole does not appear to account for the central nervous system toxicity of niridazole.

Identification and properties of myocardial myoglobin as a binder of iodothyronines.

Gel filtration of dog myocardial cytosol previously incubated with [125I]T4 or [125I]T3 revealed hormone binding in three fractions, one of which, M-2, was presumptively identified as myoglobin by absorbance maximum, molecular weight and specific immunodiffusion. Gel chromatography of purified horse or dog myoglobins incubated with labeled T3 or T4 resulted in coelution of the myoglobin and iodothyronine peaks. Excess unlabeled thyroid hormone displaced no more than 25% of tracer bound to myoglobin. Acid-acetone fractionation of myoglobin into heme and globin, and subsequent precipitation of the heme, localized hormone binding to the heme moiety. Hematin (ferric state heme) in solution was also shown to bind thyroid hormone. Added to human sera which were then subjected to T3 or T4 radioimmunoassay, myoglobin reduced detectable, endogenous iodothyronine by 77 and 26%, respectively. The myoglobin effect was concentration dependent. Heart myoglobin, like hemoglobin in the erythrocyte, is a cytoplasmic heme protein responsible for a major fraction of binding of intracellular iodothyronine. The nature of the interaction between iodothyronines and the heme prosthetic group is unclear.

Selective and potent beta 2-adrenoceptor agents within the tetrahydroisoquinoline class: effect of methyl substitution at the benzylic carbon of the 1-(3,4,5-trimethoxybenzyl) group of trimetoquinol.

A systematic series of methyl (2 and 3) and dimethyl (4) analogues of trimetoquinol (1) were synthesized and evaluated for their beta 1 (atria) and beta 2 (trachea) and adrenoceptor activities. Structural assignments for the erythro (2) and the threo (3) diastereoisomers of 1-(3,4,5-trimethoxy-alpha-methylbenzyl)-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline were based on NMR spectra of the 6,7-dibenzyl precursors 15 and 16, respectively, and on the synthetic derivatives of cis- and trans-13-methyl-2,3-bis(benzyloxy)-9,10,11-trimethoxytetrahydroprotoberberine (18 and 17). The rank order of beta 2-agonist activity for these compounds was 3 greater than 1 greater than 2 greater than 4. The rank order of activity as beta 1 agonists on the guinea pig atria is 1 greater than 3 greater than 2, and 4 was inactive. The methylated analogues show selectivity for beta 2 receptors in our preliminary pharmacological studies. The threo isomer 3 is the most potent and selective beta 2 stimulant reported to date in the tetrahydroisoquinoline class.

Relative importance of bacterial and mammalian nitroreductases for niridazole mutagenesis.

Niridazole is a nitrothiazole anthelmintic agent used to treat schistosomiasis. Its antibacterial activity was found to require the presence of the nitro group; a synthetic desnitro analog was completely inactive. Niridazole was mutagenic for Salmonella tester strains TA1538, TA98, and TA100, suggesting that it was both a frame-shift- and a base substitution-type mutagen. It was effective under both aerobic and anaerobic conditions, while similar testing of the desnitro niridazole produced consistently negative results. Addition of rat liver S-9 fraction under either aerobic or anaerobic conditions did not enhance mutagenicity. However, since bacterial killing limited the dose of niridazole to 0.33 microgram/plate in standard tester strains (1/20 Km for the mammalian liver enzymes), further studies were performed using niridazole-resistant, histidine-dependent mutants derived from strains TA98 and TA100. These mutants were found to be nitroreductase deficient and to resist the mutagenic effects of niridazole, in the presence or absence of S-9, up to concentrations of 10 microgram/plate. In addition, even at niridazole concentrations of up to 100 microgram/plate, rat liver S-9 was ineffective in enhancing the mutagenicity of niridazole. These results suggest that the mutagenicity of niridazole is dependent on its aromatic nitro group and a specific bacterial nitroreductase.