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PubMed1988

Cathepsin D and 2',3'-cyclic nucleotide 3'-phosphohydrolase in developing human foetal brain.

Three peaks of proteinases were observed with hemoglobin, bovine serum albumin and casein as substrates at the pH of 3.5, 6.5 and 8.5, in prenatal human cerebral cortex. Cathepsin D (EC 3.4.23.5) was the most prominent, with hemoglobin as the preferred substrate. The enzyme was partially purified by Concanavalin A - Sepharose affinity chromatography and the nature of the active site was assessed with proteinase inhibitors. Inhibitor studies showed that similar to pepstatin A, benzethonium chloride was also strongly inhibitory to the enzyme. The distribution of cathepsin D, a neuronal marker, and 2',3'-cyclic nucleotide 3'-phosphohydrolase (EC 3.1.4.37), a oligodendroglial marker in foetal brain regions with increasing gestation revealed that neurogenesis and gliogenesis occur concomitantly from earlier periods of gestation. Glial marker acquisition was particularly high in medulla and in spinal cord between 20 and 25 weeks of gestation.

Brain↗

PubMed2005

Problems with the estimation of urine protein by automated assays.

OBJECTIVES: Most clinical laboratories replaced their manual precipitation techniques for the determination of urinary protein with automated dye binding assays or benzethonium chloride-turbidimetric assays. Few studies have validated these assays for the measurement of urinary proteins in the normal range. DESIGN AND METHODS: This study compares four automated assays for the measurement of urinary protein to a manual Ponceau S/TCA precipitation assay. We evaluated the linearity, the precision, the analytical sensitivity, the accuracy and the recovery of different proteins for each assay. RESULTS: All assays showed good linearity with the theoretical concentration of albumin present in the sample. The coefficient of variation was below 10% at a concentration of 0.142 g/L. However, the manual Ponceau S/TCA assay demonstrated superior analytical sensitivity. Accuracy determinations showed a variable positive bias and poor correlations at concentrations below 0.1 g/L when compared to the Ponceau S/TCA assay. Small molecular weight peptides particularly affected the pyrogallol red assays but other urinary components also interfered with the automated assays. CONCLUSIONS: Most automated assays show high imprecision and poor accuracy for the measurement of urinary protein in the normal range. The Ponceau S/TCA offers a precise and accurate manual alternative to these automated assays.

Albuminuria↗

PubMed2000

Variability of standard clinical protein assays in the analysis of a model urine solution of fragmented albumin.

OBJECTIVES: This study investigates the sensitivity of various standard clinical techniques in the detection of albumin fragments. The significance of this work is in the detection of urinary proteins, such as albumin, which has recently been discovered to be excreted as mainly peptide fragments as a result of filtered albumin undergoing degradation during renal passage. All filtered proteins undergo a similar degradation process. DESIGN AND METHODS: Albumin digested with trypsin was used as a model urine solution. The solution was assayed for albumin concentration by various methods including the biuret assay that is known to detect urinary albumin fragments. The digest solution was also analyzed by various clinically used chromagen assays, electrophoretic and chromatographic methods to determine whether they are able to detect the fragmented protein. RESULTS: The benzethonium chloride, Coomassie blue, and pyrogallol red assays for urine protein, the immunoassay for human albumin and sodium dodecyl sulfate polyacrylamide gel electrophoresis with Coomassie blue staining were unable to detect the albumin fragments. Capillary electrophoresis was sensitive to the fragments but with low resolution. High-performance liquid chromatography gave the best results. CONCLUSIONS: Many techniques utilized to assay patient urine samples are unable to detect fragmented albumin and, hence, will severely underestimate albumin and protein excretion.

Albuminuria↗

PubMed2002

Displacement chromatography of isomers and therapeutic compounds.

Displacement chromatography was successfully used to separate a binary isomer mixture, epirubicin and doxorubicin, on Kromasil KR100-10 C18 250x4.6 mm I.D. (10 microm) column. Displacement parameters such as the types and the concentrations of displacer, the composition and the flow rate of the mobile phase were critically examined in this study. The displacer employed was 30 mg/ml benzethonium chloride. Loading of feed at lower initial organic level of mobile phase coupled with displacement at higher organic level was found to give efficient separation. A 30-mg amount of binary isomer mixture was separated on an analytical column. The purification of epirubicin from the closely related impurities present in raw product solution by displacement chromatography was also investigated. The purity of epirubicin required was greater than 99% with a recovery of 60%. The results have indicated that this process made good use of the high feed load, low solvent costs, and high resolution characteristics of displacement chromatography and offered the chromatographic engineer a powerful tool for the preparative purification of therapeutic compounds.

Chromatography, Liquid↗

PubMed2003

An evaluation of protein assays for quantitative determination of drugs.

We have evaluated the response of six protein assays [the biuret, Lowry, bicinchoninic acid (BCA), Coomassie Brilliant Blue (CBB), Pyrogallol Red-Molybdate (PRM), and benzethonium chloride (BEC)] to 21 pharmaceutical drugs. The drugs evaluated were analgesics (acetaminophen, aspirin, codeine, methadone, morphine and pethidine), antibiotics (amoxicillin, ampicillin, gentamicin, neomycin, penicillin G and vancomycin), antipsychotics (chlorpromazine, fluphenazine, prochlorperazine, promazine and thioridazine) and water-soluble vitamins (ascorbic acid, niacinamide, pantothenic acid and pyridoxine). The biuret, Lowry and BCA assays responded strongly to most of the drugs tested. The PRM assay gave a sensitive response to the aminoglycoside antibiotics (gentamicin and neomycin) and the antipsychotic drugs. In contrast, the CBB assay showed little response to the aminoglycosides and gave a relatively poor response with the antipsychotics. The BEC assay did not respond significantly to the drugs tested. The response of the protein assays to the drugs was further evaluated by investigating the linearity of the response and the combined response of drug plus protein. The results are discussed with reference to drug interference in protein assays and the development of new methods for the quantification of drugs in protein-free solution.

Analgesics↗

PubMed2005

Urine protein estimation in hypertensive pregnancy: which thresholds and laboratory assay best predict clinical outcome?

OBJECTIVE: To determine what threshold for proteinuria could best predict clinical outcome and whether this threshold could be applied universally to any biochemical assay. DESIGN: A prospective observational study of hypertensive pregnancies referred for further assessment after in a UK University hospital (n=197). Twenty-four hour urine protein was measured by two different assays [benzethonium chloride assay (BCA) and Bradford assay]. The differences between the two assays were calculated from Receiver Operating Characteristic (ROC) curves. Commonly used thresholds for defining preeclampsia (0.3 and 0.5 g/24 hours) were explored for both assays for the prediction of adverse clinical outcomes (severe hypertension, Birthweight<10th percentile, preterm delivery, and a composite biochemical/haematological derangement). RESULTS: The two assays are not equivalent. The prevalence of>300 mg/24 hour proteinuria and, hence, the prevalence of preeclampsia differed between the two assays. ROC curve analysis demonstrates that the two assays are similar in terms of overall performance as predictive tests. However the threshold of 300 mg/24 hours performs poorly as a predictor of clinical risk. Likelihood ratios (LR) for the BCA at the 300 mg/L threshold for each clinical outcome do not achieve statistical significance. At the 500 mg/L threshold, the LR+for the BCA assay does achieve statistical significance for severe hypertension (LR+:1.51 95% CI 0.99-2.28) and for birthweight<10th percentile (LR+:1.72 95% CI 1.11-2.66). For the Bradford assay at the 300 mg/24 hour threshold, the LR+does achieve statistical significance for birthweight<10th percentile (LR+:1.71 95% CI 1.41-4.31). However, at the 500 mg/24 hour threshold, the LR+is significant for severe hypertension (LR+:2.15 95% CI 1.07-4.34), birthweight<10th percentile (LR+:2.79 95% CI 1.4-5.54) and biochemical disease (LR+:2.47 95% CI 1.22-5.01). CONCLUSIONS: This study suggests that thresholds for proteinuria need to be higher (possibly>or=0.5 g/24 hours) and there is the need for a "gold standard" proteinuria assay against which all other measures of quantification can be assessed.

Adult↗

PubMed1998

A new approach to identifying genotoxic carcinogens: p53 induction as an indicator of genotoxic damage.

The tumor suppressor gene p53 encodes a nuclear phosphoprotein which is critical for cell cycle control and prevention of uncontrolled cell proliferation that can lead to cancer. Previous studies have shown that cells respond to DNA damage by increasing their levels of p53, which then acts to prevent replication of damaged DNA. This study examined the effects on p53 protein levels of several different categories of chemical carcinogens. N-Methyl-N'-nitro-nitrosoguanidine and N-ethyl-N-nitrosourea, two direct-acting genotoxic (DNA-reactive) carcinogens, caused p53 induction as early as 2 h following treatment, with peak increases within 4-12 h. Aflatoxin B1 and 2-acetylaminofluorene, indirect-acting genotoxic carcinogens, caused a later induction of p53, with the peak increase appearing between 16 and 24 h following treatment. These observations demonstrate a correlation between p53 induction pattern and DNA damaging mechanism of genotoxins. Phenol, diethylstilbestrol and ethylacrylate also induced increases in cellular p53. The half-life of p53 protein was increased in cells treated with genotoxic agents. On the other hand, the epigenetic (non-DNA-reactive) carcinogens azathioprine and saccharin, as well as two substances generally considered to be non-carcinogens, dimethylsulfoxide and benzethonium chloride, had no effect on p53 protein levels of treated cells. Measurement of the cytotoxic effects of each of these chemicals led to the conclusion that p53 protein induction is not a general, non-specific consequence of the cytotoxic effect of these genotoxins. These results suggest that measurement of p53 protein induction may be an effective tool to identify environmental genotoxins.

Acrylates↗

PubMed1999

Development of a transgenic mouse model for carcinogenesis bioassays: evaluation of chemically induced skin tumors in Tg.AC mice.

Transgenic rodent models have emerged as potentially useful tools in the assessment of drug and chemical safety. The transgenic Tg.AC mouse carries an inducible v-Ha-ras oncogene that imparts the characteristic of genetically initiated skin to these animals. The induction of epidermal papillomas in the area of topically applied chemical agents, for duration of not more than 26 weeks, acts as a reporter phenotype that defines the activity of the test article. We describe here the activity of six chemicals that have been previously characterized for activity in the standard 2-year bioassay conducted by the National Toxicology Program (NTP). Homozygous female Tg.AC mice were treated with benzene (BZ), benzethonium chloride (BZTC), o-benzyl-p-chlorophenol (BCP), 2-chloroethanol (2-CE), lauric acid diethanolamine (LADA) and triethanolamine (TEA). BZ and LADA induced skin papillomas in a dose-dependent manner, while BCP induced papillomas only at the highest dose. BZTC, 2-CE, and TEA exhibited no activity. The correspondence of chemical activity in Tg.AC mice with that in the 2-year bioassay was high. A comparison of responsiveness to BZ and LADA was made between hemizygous and homozygous female Tg.AC mice. Both genotypes appear to be equally sensitive to maximum doses of active compounds. The results reported here indicate that the Tg.AC transgenic mouse model can discriminate between carcinogens and noncarcinogens and that both mutagenic and nonmutagenic chemicals can be detected. These studies provide support for the adjunctive use of the Tg.AC transgenic mouse skin tumor model in drug and chemical safety assessment and for the prediction of the carcinogenic potential of chemicals.

Animals↗

PubMed2005

Susceptibilities to antiseptic agents and distribution of antiseptic-resistance genes qacA/B and smr of methicillin-resistant Staphylococcus aureus isolated in Asia during 1998 and 1999.

Many antiseptic agents are used in hygienic handwashes in the prevention of nosocomial infections by methicillin-resistant Staphylococcus aureus (MRSA). The plasmid-borne genes qacA/B and smr confer resistance to cationic antiseptic agents in S. aureus. In this study, the susceptibilities for dyes and antiseptic agents (e.g. acriflavine, acrinol, benzalkonium chloride, benzethonium chloride, chlorhexidine digluconate and alkyldiaminoethylglycine hydrochloride) of 894 isolates of MRSA collected from 11 Asian countries (South Korea, China, the Philippines, Singapore, Vietnam, Thailand, Indonesia, India, Sri Lanka, Saudi Arabia and Japan) between 1998 and 1999 were examined. In addition, the distributions of the antiseptic-resistance genes qacA/B and smr were studied by PCR. Among the Asian MRSA isolates 57.7% (516/894) were acriflavine-resistant. The smr gene was detected in 31.6% (12/38) of MRSA isolates from India but only in 1.9% (16/856) of all the isolates from other Asian countries. MRSA with qacA/B comprised 41.6% (372/894) of the isolates across Asia. In addition, PFGE was performed to type the MRSA and grouped the tested 30 MRSA isolates with qacA/B into 21 PFGE types. The results indicated that qacA/B is functionally the most important gene mediating antiseptic resistance in the MRSA strains of Asia and that a specific MRSA with qacA/B was not prevalent in Asia but qacA/B were widely spread among MRSA of Asia, while the geographical distribution of smr is more limited.

Acriflavine↗

PubMed1995

Effect on neonatal thyroid function of povidone-iodine used on mothers during perinatal period.

OBJECTIVE: Our objective was to evaluate the effect on the neonatal thyroid function of povidone-iodine (PVP-I) used on mothers during the perinatal period. METHODS: Eight consecutive-term pregnant women were randomly separated into 4 groups. Either PVP-1 or benzethonium chloride (BC) was used to disinfect both maternal skin during labor and vaginal lacerations after delivery in Group I and IV; and PVP-1 or BC was supplied to either skin or lacerations in Groups II and III. On day 5, we measured the iodide concentration in breast milk and random urine of neonates, and the levels of neonatal thyroid-stimulating hormone (TSH) and free thyroxine. RESULTS: The TSH levels in Group I and the breast-milk iodide concentrations in Groups I and II were significantly higher than those in Group IV (p < 0.05). Dietary iodide intake from breast milk correlated significantly with urinary iodide concentrations (r = 0.52, p < 0.001). In our clinic, the recall rate at the time of screening for congenital hypothyroidism was significantly decreased by discontinuing the use of PVP-I (4.47% vs 0.74%, p < 0.001). CONCLUSIONS: These findings suggest that the use of PVP-I on mothers is associated with transient neonatal hyperthyrotropinemia, in which breast milk might play an important role. Therefore, we recommend that the use of PVP-I be avoided during labor.

Anti-Infective Agents, Local↗

PubMed1984

Development of topical treatment for cutaneous leishmaniasis caused by Leishmania major in experimental animals.

Topical treatment, with drug-containing ointments, of cutaneous leishmaniasis caused by Leishmania major in BALB/c mice was studied. Twenty chemotherapeutic agents having potential or established antileishmanial activity were formulated in different ointment and cream bases. Only 15% paromomycin sulfate with 12% methylbenzethonium chloride, 12% benzethonium chloride, 12% cetalkonium chloride, or 12% dimethyl sulfoxide, all incorporated in white soft paraffin (United Kingdom patent application no. 2117237A), were completely effective. Topical treatment twice daily for 6 or more days caused total elimination of the parasites and healing of the lesion in all treated mice. All the other antileishmanial compounds, including sodium stibogluconate, pentamidine, amphotericin B, emetine hydrochloride, metronidazole, co-trimoxazole, allopurinol, and rifampin, either showed a slight effect on the parasites or were highly toxic to the animal host at the concentrations tested.

Administration, Topical↗

PubMed1963

Studies on immunity in anthrax. X. Gel-adsorbed protective antigen for immunization of man.

Puziss, Milton (Fort Detrick, Frederick, Md.) and George G. Wright. Studies on immunity in anthrax. X. Gel-adsorbed protective antigen for immunization of man. J. Bacteriol. 85:230-236. 1963.-Protective antigen in filtrates of anaerobic cultures of Bacillus anthracis was not readily precipitable by alum or other aluminum salts, but was adsorbed onto a preformed aluminum hydroxide gel under specific conditions. The adsorbed product was highly effective in immunizing rabbits, guinea pigs, and mice against challenge with virulent B. anthracis spores. Gel-adsorbed anaerobic antigens preserved with 1:10,000 thimerosal were unstable on storage at 4 C; replacement of this preservative with 1:40,000 benzethonium chloride produced a more stable product. Addition of 0.0009% formalin further increased the stability during accelerated aging at 37 C. The final product had high protective activity in animals and was well tolerated in man.

Alum Compounds↗

PubMed1992

Relationship between the effects on bacterial activity of selected disinfectants and the hydrophobic characters of dibasic acid diesters.

We prepared test solutions which contained 80% (v/v) ethanol and 0.2% (w/v) chlorhexidine (CH) or benzalkonium chloride (BC) with or without a dibasic acid diester. After complete evaporation of the ethanol from the solution on filter paper, an overnight broth culture (Staphylococcus aureus) was repeatedly inoculated onto the filter paper, and viable bacterial counts were measured at 5 min after the last inoculation. By comparison with viable counts for CH or BC alone, we estimated the potentiating effects of dibasic acid diester on the bactericidal activity of CH or BC, and confirmed that this activity of the two disinfectants was potentiated in the presence of certain compounds in the homologs of di-n-butyl esters of aliphatic dibasic acid, and di-alkyl esters of adipic and phthalic acid. Diisobutyl adipate, one of the most effective diesters, substantially enhanced the bactericidal activities of benzethonium chloride, cetyl pyridinium chloride and didecyl dimethyl ammonium chloride, as well as CH and BC, but not those of polyhexamethylene biguanide or alkyldiaminoethyl glycinate. The potentiating effects of dibasic acid diesters observed for both CH and BC seemed to be affected by the hydrophobic character of these diesters themselves and are also expressed well by a particular quadratic equation as a function of these characters: namely, capacity factors, as determined by high-performance liquid chromatography.

Chemical Phenomena↗

PubMed2002

Characteristics of hyaluronate-hydroxyethyl acrylate blend gel and release of cationic amphiphilic solutes.

Hyaluronate-hydroxyethyl acrylate blend gel (HA-PHEA) were prepared to modify the brittleness of hyaluronate gel (HA) and the characteristics of HA-PHEA gel were compared with those of HA and polyhydroxyethyl acrylate (PHEA) gels. These gels were high in water content and transparent. HA-PHEA gel was improved in viscoelastic properties due to the elasticity and the high affinity with water of PHEA, and the drying-swelling cycles became reversible. The effective charge densities theta of the gels estimated from membrane potentials were -0.002, -0.008 and 0 mol dm(-3) for HA-PHEA, HA and PHEA gels. Effects of electro- static and nonelectrostatic interactions on absorptions and releases were studied using sodium benzoate (NaBA) as an anionic solute, and methylene blue (MB), chlorpromazine (CPHCl) and benzethonium chloride (BZTCl) as cationic solutes, in which CPHCl and BZTCl are cationic amphiphilic solutes. The releases of MB, CPHCl and BZTCl from HA-PHEA and HA gels were suppressed comparing with those of NaBA. By adding salts, the releases of MB and CPHCl were enhanced but those of BZTCl were suppressed due to enhancement of the intra- and intermicelle formation. In the releases of the cationic solutes from HA-PHEA gel, electrostatic and nonelectrostatic interactions with HA were found to play important roles. Behaviors of the releases from HA-PHEA gel were found to possess the features of HA gel.

Acrylates↗

PubMed2005

[Relation between oxygen uptake rate and biosorption of activated sludge against chemical substance].

In this study, the elucidation of the toxicity mechanism was undertaken regarding the IC(50) of the oxygen uptake rate (OUR) with relevance to the biosorption as a toxicity evaluation of chemical substances for activated sludge (AS). At the IC(50) of<100 mg/l, malachite green (MG) and crystal violet (CV) were confirmed in the group showing relatively strong OUR inhibition. These dyes were markedly biosorbed by AS in a short time. The biosorption for AS showed a weak tendency in linear alkyl benzene sulfonate (LAS), alkyl ethoxy sulfonate (AES), alpha-olefine sulfonate (AOS), sodium dodecyl sulfate (SDS), formaldehyde (FA), benzalkonium chloride (BZaC), benzethonium chloride (BZeC), rhodamine 6G (R-6G) and fuchsine (Fuc) in which the IC(50) belonged to the 100-1000 mg/l group, when it was compared with CV and MG. In ethanol (EtOH), isopropanol (PrOH), nile blue (NB), evans blue (EB), methylene blue (MB), methyl orange (MO), paraquat (PQ), chlorophyllin (Chl) and auramine (Aur), the IC(50) was large, and the biosorption of AS was weak at 0-15%. The biosorption of MG for AS followed the adsorption isotherm equation Y=0.002X(0.511) of Freundrich. The correlation coefficient was gamma=0.998 (n=8), and a very high correlation was obtained. In the qualitative OUR curve by AS pretreated with MG or CV which belonged to the IC(50) small group, the inhibition of remarkable OUR was observed. Therefore, the findings of the present investigation suggest that the inhibition of the OUR for AS by the tested chemical substances was markedly affected by the biosorption.

Adsorption↗

PubMed2005

Ability of fourteen chemical agents used in dental practice to induce chromosome aberrations in Syrian hamster embryo cells.

To assess the genotoxicity of 14 chemical agents used in dental practice, the ability of these agents to induce chromosome aberrations was examined using Syrian hamster embryo (SHE) cells. Statistically significant increases in the frequencies of chromosome aberrations were induced in SHE cells treated with 7 of 10 chemical agents used as endodontic medicaments, that is, carbol camphor, m-cresol, eugenol, guaiacol, zinc oxide, hydrogen peroxide, and formaldehyde. The other 3 chemical agents, that is, thymol, glutaraldehyde, and iodoform, did not increase the levels of chromosome aberrations. Of the 4 chemical agents that are used as an antiseptic on the oral mucosa, chromosome aberrations were induced by iodine, but not by the other 3 antiseptics, benzalkonium chloride, benzethonium chloride, and chlorhexidine. Among the 6 chemical agents exhibiting a negative response in the assay, only thymol induced chromosome aberrations in the presence of exogenous metabolic activation. Our results indicate that chemical agents having a positive response in the present study are potentially genotoxic to mammalian cells and need to be studied further in detail.

Animals↗

PubMed2002

Burns caused by dilute hydrofluoric acid in the bleach.

Two male cleaning workers aged 62 (patient 1) and 28-(patient 2) presented with red, swollen, aching hands and fingers. At the first interview, the fingers of the right hand of both patients were swollen from the proximal interphalangeal joint to the tip of the finger. The fingers were red and intensely painful. The bleach that both patients used contained 9.5% hydrofluoric acid, and therefore we diagnosed their symptoms as those of burns caused by this agent. We subcutaneously injected 8.5% calcium gluconate into the affected fingers and dressed them with gauze soaked in cooled 0.025% benzethonium chloride. The patients did not use gloves at work, neither of them knew that the cleaning fluid contained dilute hydrofluoric acid, and they were unaware of the danger of this agent. They had not received proper education about the care and handling of poisons and deleterious substances such as hydrofluoric acid. The doctors who had examined the patients in the emergency ward overlooked the possibility of hydrofluoric acid burns, although they suspected chemical burns and confirmed the trade name of the cleaning agent. In addition, although the patients presented with intense pain, no white areas of coagulation and blistering, or surrounding erythemas, which are characteristic symptoms of such burns, were evident. The component of the cleaning agent was described on the container label in very small print. Had the words "poison" and "hydrogen fluoride" been printed in large characters, the examining doctors in the emergency ward would probably not have overlooked the presence of hydrofluoric acid, and the patients would perhaps have been more careful when using it. Hydrofluoric acid can be easily obtained by anyone through the Internet, although general consumers could not obtain industrial quantities. Therefore, the number of burn patients who are not familiar with hydrofluoric acid may increase in the future.

Adult↗

PubMed1987

Inactivation of Bacillus piliformis spores by heat and certain chemical disinfectants.

The inactivation of Tyzzer's organism (Bacillus piliformis) spore isolated from rats by heat and various chemical disinfectants was studied. The spores were from B. piliformis-infected rat liver tissues. The spore suspension (10(4) 50% of rat liver lesion producing dose with prednisolone treatment/ml) was treated with heart or disinfectants. Inactivation of the spores was examined in experimentally infected rats. Rats were inoculated perorally with a treated spore suspension and injected subcutaneously with prednisolone. On the sixth day after inoculation, rats were examined grossly for liver lesions. Spores were inactivated at 80 degrees C for 15 min but not at 60 degrees C for 30 min. Spores were inactivated by 0.4% peracetic acid, 0.015% sodium hypochrolite, 1% iodophol, 5% phenol. Alcide and 0.37% formaldehyde solution, but not by 0.037% formaldehyde solution, 70% ethanol, 0.3% benzethonium chloride solution, 3% cresol and soap solution, or 4% chlorhexidine digluconate. These findings suggest that B. piliformis spores are relatively sensitive to heat and certain chemical disinfectants.

Animals↗