Alcohol stress on cyanobacterial membranes: New insights revealed by transcriptomics.

Cyanobacteria are model photosynthetic prokaryotic organisms often used in biotechnology to produce biofuels including alcohols. The effect of alcohols on cyanobacterial cell physiology and specifically on membrane fluidity is poorly understood. Previous research on various primary aliphatic alcohols found that alcohols with a short hydrocarbon chain (C1-C3) do not affect expression of genes related to membrane physical state. In addition, less water-soluble alcohols with a hydrocarbon chain longer than C8 are found to have a reduced ability to reach cellular membranes hence do not drastically change membrane physical state or induce expression of stress-responsive genes. Therefore, hexan-1-ol (C6) is suggested to have the most profound effect on cyanobacterial membrane physical state. Here, we studied the effects of hexan-1-ol on the cyanobacterium Synechocystis sp. PCC 6803 transcriptome. The transcriptome data obtained is compared to the previously reported analysis of gene expression induced by benzyl alcohol and butan-1-ol. The set of genes whose expression is induced after exposure to all three studied alcohols is identified. The expression under alcohol stress for several general stress response operons is analyzed, and examples of antisense interactions of RNA are investigated.

Safety Assessment of Benzyl Alcohol, Benzoic Acid and its Salts, and Benzyl Benzoate.

Benzyl alcohol, benzoic acid and its salts, and benzyl benzoate function mostly as fragrance ingredients/preservatives in cosmetic products. The Cosmetic Ingredient Review Expert Panel previously established concentration limits for benzyl alcohol, benzoic acid, and sodium benzoate in cosmetics and determined that the available data were insufficient to support the safety of these ingredients during inhalation exposure. After reviewing newly available data, it was concluded that benzyl alcohol, benzoic acid and its salts, and benzyl benzoate are safe in the present practices of use and concentration described in this safety assessment.

Sulfation of benzyl alcohol by the human cytosolic sulfotransferases (SULTs): a systematic analysis.

The aim of the present study was to identify human cytosolic sulfotransferases (SULTs) that are capable of sulfating benzyl alcohol and to examine whether benzyl alcohol sulfation may occur in cultured human cells as well as in human organ homogenates. A systematic analysis revealed that of the 13 known human SULTs, SULT1A1 SULT1A2, SULTA3, and SULT1B1 are capable of mediating the sulfation of benzyl alcohol. The kinetic parameters of SULT1A1 that showed the strongest benzyl alcohol-sulfating activity were determined. HepG2 human hepatoma cells were used to demonstrate the generation and release of sulfated benzyl alcohol under the metabolic settings. Moreover, the cytosol or S9 fractions of human liver, lung, kidney and small intestine were examined to verify the presence of benzyl alcohol sulfating activity in vivo. Copyright © 2015 John Wiley & Sons, Ltd.

Potential confounding effects of benzyl alcohol as a formulation excipient support the elimination of the abnormal toxicity test from pharmacopoeias.

Benzyl alcohol is an excipient used in many drugs as a stabilizer. Depending on the amount present in drug formulations there might be confounding findings in the Abnormal Toxicity Test (ATT). The ATT is utilized as a quality control (QC) release test to detect extraneous contaminants according to national pharmacopoeias. Our study assessed the effects of benzyl alcohol as defined in ATT designs. This study - the first thorough evaluation of the confounding effects of benzyl alcohol on the ATT - was conducted in relation to particular health authority questions and was part of the root-cause analyses resulting from some transient behavioral findings observed in the test. Two strains of mice, CD-1 & Kunming, plus Hartley guinea pigs were administered intraperitoneally (ip), subcutaneously (sc), or intravenously (iv) with benzyl alcohol at dose level defined in the ATT design. In both mice and guinea pigs, only after ip administration, minimal behavioral changes were observed transiently within 2-3 min after administration. Therefore, the presence of benzyl alcohol in the product batch may confound the ATT results. This study provides further evidence to question the validity of the ATT for its intended use.

Co-exposure with fullerene may strengthen health effects of organic industrial chemicals.

In vitro toxicological studies together with atomistic molecular dynamics simulations show that occupational co-exposure with C60 fullerene may strengthen the health effects of organic industrial chemicals. The chemicals studied are acetophenone, benzaldehyde, benzyl alcohol, m-cresol, and toluene which can be used with fullerene as reagents or solvents in industrial processes. Potential co-exposure scenarios include a fullerene dust and organic chemical vapor, or a fullerene solution aerosolized in workplace air. Unfiltered and filtered mixtures of C60 and organic chemicals represent different co-exposure scenarios in in vitro studies where acute cytotoxicity and immunotoxicity of C60 and organic chemicals are tested together and alone by using human THP-1-derived macrophages. Statistically significant co-effects are observed for an unfiltered mixture of benzaldehyde and C60 that is more cytotoxic than benzaldehyde alone, and for a filtered mixture of m-cresol and C60 that is slightly less cytotoxic than m-cresol. Hydrophobicity of chemicals correlates with co-effects when secretion of pro-inflammatory cytokines IL-1ß and TNF-α is considered. Complementary atomistic molecular dynamics simulations reveal that C60 co-aggregates with all chemicals in aqueous environment. Stable aggregates have a fullerene-rich core and a chemical-rich surface layer, and while essentially all C60 molecules aggregate together, a portion of organic molecules remains in water.

Airborne exposures to monoethanolamine, glycol ethers, and benzyl alcohol during professional cleaning: a pilot study.

UNLABELLED: A growing body of epidemiologic evidence suggests an association between exposure to cleaning products and respiratory dysfunction. Due to the lack of quantitative assessments of respiratory exposures to airborne irritants and sensitizers among professional cleaners, the culpable substances have yet to be identified. PURPOSE: Focusing on previously identified irritants, our aims were to determine (i) airborne concentrations of monoethanolamine (MEA), glycol ethers, and benzyl alcohol (BA) during different cleaning tasks performed by professional cleaning workers and assess their determinants; and (ii) air concentrations of formaldehyde, a known indoor air contaminant. METHODS: Personal air samples were collected in 12 cleaning companies, and analyzed by conventional methods. RESULTS: Nearly all air concentrations [MEA (n = 68), glycol ethers (n = 79), BA (n = 15), and formaldehyde (n = 45)] were far below (<1/10) of the corresponding Swiss occupational exposure limits (OEL), except for ethylene glycol mono-n-butyl ether (EGBE). For butoxypropanol and BA, no OELs exist. Although only detected once, EGBE air concentrations (n = 4) were high (49.48-58.72mg m(-3)), and close to the Swiss OEL (49mg m(-3)). When substances were not noted as present in safety data sheets of cleaning products used but were measured, air concentrations showed no presence of MEA, while the glycol ethers were often present, and formaldehyde was universally detected. Exposure to MEA was affected by its amount used (P = 0.036), and spraying (P = 0.000) and exposure to butoxypropanol was affected by spraying (P = 0.007) and cross-ventilation (P = 0.000). CONCLUSIONS: Professional cleaners were found to be exposed to multiple airborne irritants at low concentrations, thus these substances should be considered in investigations of respiratory dysfunctions in the cleaning industry; especially in specialized cleaning tasks such as intensive floor cleaning.

Further characterization of ocular safety profile of commercially available preserved and preservative-free triamcinolone acetonide.

PURPOSE: To characterize the safety profile of triamcinolone acetonide (TA) for intraocular application. METHODS: In vitro cell viability assay was performed on 2 types of human ocular cells to evaluate the cytotoxicity of the simulated different vitreal concentrations (from a 1:15 dilution as if injected into 1.5 mL of rabbit vitreous to 1:50 dilution as if injected into 5 mL of human vitreous) of preservative and excipient (supernatant) from Kenalog-40. In vivo 35 guinea pigs were used for evaluating either a dose of intravitreal triamcinolone acetonide (Kenalog-40 and Triesence) or the supernatant of Kenalog-40. The animal eyes were monitored by biomicroscopy, ophthalmoscopy, tonometry, electroretinography, and histology. RESULTS: A ≥ 1:15 dilution of triamcinolone acetonide supernatant from Kenalog-40 did not show cytotoxicity on cultured human pigment epithelial (retinal pigment epithelium) cells or Müller cells. In vivo, neither intravitreal 6 µL (0.248 mg, equivalent to 4 mg in 0.1 mL for human eyes) nor 18 µL (0.744 mg, equivalent to 4 mg in 0.1 mL for rabbit eyes and equivalent to 12 mg in 0.1 mL for human eyes) of triamcinolone acetonide suspension showed ocular toxicity. No significant difference was noted between Kenalog-40 and Triesence clinically and histopathologically. CONCLUSION: The equivalent triamcinolone acetonide doses to 0.1 mL (4 or 12 mg) intravitreal injection for human eye were found safe in guinea pig eyes. No significant difference was noted for 0.1 mL intravitreal injection between Kenalog-40 and Triesence.

Fragrance material review on benzyl alcohol.

A toxicologic and dermatologic review of benzyl alcohol when used as a fragrance ingredient is presented. Benzyl alcohol is a member of the fragrance structural group Aryl Alkyl Alcohols and is a primary alcohol. The AAAs are a structurally diverse class of fragrance ingredients that includes primary, secondary, and tertiary alkyl alcohols covalently bonded to an aryl (Ar) group, which may be either a substituted or unsubstituted benzene ring. The common structural element for the AAA fragrance ingredients is an alcohol group -C-(R1)(R2)OH and generically the AAA fragrances can be represented as an Ar-C-(R1)(R2)OH or Ar-Alkyl-C-(R1)(R2)OH group. This review contains a detailed summary of all available toxicology and dermatology papers related to this individual fragrance ingredient and is not intended as a stand-alone document. Available data for benzyl alcohol were evaluated then summarized and includes physical properties, acute toxicity, skin irritation, mucous membrane (eye) irritation, skin sensitization, elicitation, phototoxicity, photoallergy, toxicokinetics, repeated dose, reproductive toxicity, genotoxicity, and carcinogenicity data. A safety assessment of the entire Aryl Alkyl Alcohols will be published simultaneously with this document; please refer to Belsito et al. (2012) for an overall assessment of the safe use of this material and all Aryl Alkyl Alcohols in fragrances.

Mechanisms underlying benzyl alcohol cytotoxicity (triamcinolone acetonide preservative) in human retinal pigment epithelial cells.

PURPOSE: Benzyl alcohol (BA) is the preservative in triamcinolone acetonide (TA) suspensions, which are used in treating vitreoretinal diseases and during surgery. This paper investigates the molecular mechanisms and signaling pathways underlying BA toxicity in human retinal pigment epithelial (RPE) cells. METHODS: Cultured human RPE cells from the ARPE-19 cell line were exposed to culture medium alone (control) or with BA (0.0225, 0.225, 0.9, 3, or 9 mg/mL) for up to 6 hours. BA toxicity was assessed by TUNEL assay, propidium iodide/annexin V-FITC staining and flow cytometry, caspase activation assay, caspase and apoptosis inhibition assays, mitochondrial transmembrane potential by rhodamine staining and flow cytometry, reactive oxygen species by chemiluminescence, and apoptosis-inducing factor staining. RESULTS: BA caused RPE cell death not only by necrosis but also by apoptosis, evidenced by exposure to 9 mg/mL BA for 6 hours leading to 19.0% early apoptotic cells and 64.2% apoptotic necrotic cells. Apoptotic signaling involved the immediate production of reactive oxygen species, activation of caspase-8, impairment of the mitochondrial transmembrane potential, and further activation of caspase-9 and -3. In addition, BA induced translocation of apoptosis-inducing factor into the nucleus, indicating caspase-independent apoptosis. CONCLUSIONS: BA leads to necrosis of RPE cells and triggers mitochondrial apoptosis through both caspase-dependent and - independent pathways. Extreme caution is suggested in the intraocular use of TA suspensions and meticulous evaluation before adoption of BA as a preservative in the future development of ophthalmic formulations.

[Indoor air guide values for benzyl alcohol]. / Richtwerte für Benzylalkohol in der Innenraumluft. Mitteilung der Ad-hoc-Arbeitsgruppe Innenraumrichtwerte der Innenraumlufthygiene-Kommission des Umweltbundesamtes und der Obersten Landesgesundheitsbehörden.

The German Working Group on Indoor Air Guidelines of the Federal Environment Agency and the States' Health Authorities is issuing indoor air guide values to protect public health. For health evaluation of benzyl alcohol in indoor air valid inhalation studies are missing. Following subchronic oral exposure to 800 mg benzyl alcohol/kg body weight and day neurotoxicity was observed in rats. Accounting for data gaps by a factor of 2 and extrapolating to continuous exposure by applying a factor of 2 the lowest adverse effect level for chronic exposure (LAELchron) is assessed as 200 mg benzyl alcohol/kg b. w. and day. By applying an interspecies factor of 10, an intraspecies factor of 10, a path-to-path-extrapolation and a modifying factor of 2 referring to the special physiology of children (higher breathing rate compared to adults) a health hazard guide value (RW II) of 4 mg benzyl alcohol/m3 indoor air and a health precaution guide value (RW I) of 0.4 mg benzyl alcohol/m3 are derived.

Assessment of genotoxic effects of benzyl derivatives by the comet assay.

In this study, different concentrations of four benzyl derivatives (benzyl alcohol, benzyl acetate, benzoic acid and benzaldehyde) used as flavour ingredients were investigated for genotoxicity in in vitro. By taking blood from two healthy people comet assay was carried on to investigate the potential health damages of benzyl derivatives. For the evaluation of genotoxic effects, the tail moment and % tail DNA in the treated chemicals were compared to the solvent control, which is distilled water. The alkaline comet assay showed significantly increased tail moment and % tail DNA at 25 and 50 mM concentrations of benzyl alcohol. Benzyl acetate increased both % tail DNA and tail moment at 50 mM concentrations. While % tail DNA was statistically increased at 10 mM and higher concentrations, tail moment has significant difference at 10 and 25 mM concentrations of benzaldehyde. Benzoic acid has apoptotic effects at the concentrations higher than 5 mM, for this reason we tested concentrations less than 5mM (0.05, 0.1, 0.5, 1 and 5 mM). Only the highest concentration of benzoic acid increased both tail moment and % tail DNA.

A comparative study of leukaemia inhibitory factor and interleukin-1alpha intracellular content in a human keratinocyte cell line after exposure to cosmetic fragrances and sodium dodecyl sulphate.

According to European laws animal testing in cosmetic industry will be prohibited in a few years and it will be replaced by alternative methods based on cell and tissue culture. Many ingredients of cosmetic formulations are potentially causes of skin inflammation and sensibilization. Since cytotoxicity is known, among other factors, to trigger irritation, in an alternative model for evaluation of skin irritation, it can be considered also the precocious release of inflammatory mediators, i.e. cytokines, originating mainly from keratinocytes. In this in vitro study we have analysed some parameters directly or indirectly related to irritation/inflammation, in NCTC 2544 human keratinocytes during short-time exposure to some potential irritants cosmetic fragrances, included in the European Laws 2003/15/EEC. IIC50 was extrapolated by MTT and NRU viability indexes after exposure of cell ultures to Geraniol Limonene and Benzylic Alcohol for 1, 3 and 6h. NCTC cells were then exposed to sub-toxic doses of selected compounds and interleukin-1alpha (IL-1alpha) and leukaemia inhibitory factor (LIF) expressions were analysed as early proinflammatory cytokines. To our knowledge our findings demonstrated for the first time that NCTC cells synthesize and modulate LIF after exposure to selected irritating stimuli. Moreover, our results give evidence on LIF role as in vitro precocious endpoint for the assessment of the risk in cosmetic field, because its response under irritation stimuli is very quick and comparable to IL-1alpha.

Vehicle used for triamcinolone acetonide is toxic to ocular tissues of the pigmented rabbit.

PURPOSE: To investigate the potential toxicity of the vehicle used for triamcinolone acetonide (TA) to the cornea, lens, ciliary body, and retina of pigmented rabbits. METHODS: Forty chinchilla rabbits (40 eyes) were divided into four groups: group A (control group) eyes received an intravitreal injection of 0.1 ml sterile saline solution; group B eyes received 0.1 ml (1.3 mg) TA plus vehicle; group C eyes received 0.1 ml (1.3 mg) TA alone (vehicle eliminated); group D eyes received 0.1 ml vehicle only. Intraocular pressure (IOP) was measured pre-injection and at 1, 7, 14, 30, and 90 days post-injection. Scotopic and photopic electroretinograms (ERG) were examined pre-injection and 7, 30, and 90 days post-injection. Animals were sacrificed 7, 30, and 90 days post-injection. Eyes were enucleated and examined by light microscopy (LM) and electron microscopy (EM). RESULTS: The IOP of groups B and C was higher than that of other groups on days 1, 7, and 14 post-injection (p < 0.05). ERG amplitudes of groups B and D were lower than those of other groups on days 7 and 30 post-injection (p < 0.05). Histopathological sections indicated morphologic changes in the ciliary body, lens, and retina of eyes in groups B and D. CONCLUSIONS: Vehicle used for TA is toxic to the lens, ciliary body, and retina of pigmented rabbit eyes after injection of intravitreal TA.

In vitro benzyl alcohol cytotoxicity: implications for intravitreal use of triamcinolone acetonide.

The aim of the study was to investigate the toxicity of benzyl alcohol (BA), the preservative in commercial triamcinolone acetonide (TA) suspensions, on retinal pigment epithelial (RPE) cells. Cultured RPE cells from a human cell line (ARPE-19) and from rabbits were exposed to the balanced salt solution (control) or BA (0.0225, 0.225, 0.9, 3 or 9mg/mL) for 5, 30, 60, or 120min. Morphological changes of RPE cells were evaluated by the trypan blue in situ staining. The proportions of dead cells were quantitatively measured by the trypan blue exclusion assay, and those of functional cells were assessed by a mitochondrial dehydrogenase assay. The mechanism of cytotoxicity was determined by the acridine orange/ethidium bromide staining and DNA laddering technique. Furthermore, ultrastructural changes were observed by transmission electron microscopy. The results showed that RPE cell damage was dose- and time-dependent. BA 0.225mg/mL, the clinically relevant concentration in TA following intravitreal injection, caused ultrastructural damage and impaired human RPE cell function at 2h; but BA 0.0225mg/mL did not. BA 9.0mg/mL, the concentration in commercial TA suspensions, was toxic within 5min on each assay for both human and rabbit RPE cells. The major mechanism of cell death was necrosis. In conclusion, BA in commercial TA suspensions injected intravitreally (0.225-9mg/mL) can damage RPE cells. Our in vitro study on benzyl alcohol cytotoxicity has significant clinical implications for intravitreal use of TA. We suggest that, before a commercial TA solution is used intravitreally, the vehicle should be removed to prevent damaging the RPE layer, particularly during macular hole surgery. Commercial development of a preservative-free TA suspension for intraocular use is urged.

A moephologic study of retinal toxicity induced by triamcinolone acetonide vehicles in rabbit eyes.

PURPOSE: To evaluate the toxic effects of two triamcinolone acetonide (TA) vehicles on rabbit retina at different volumes. METHODS: Vehicle A and B were prepared from two TA injections by centrifugation. Thirty-six New Zealand white rabbits were intravitreally injected with vehicle A, B, or balanced saline solution at 0.1 mL or 0.2 mL respectively. The eyes were examined by indirect ophthalmoscope, light microscope, and transmission electron microscope up to week 8 postinjection. RESULTS: Eyes with vehicle A appeared normal under the ophthalmoscope, but showed disorganization in retinal inner nuclear layer and photoreceptor layer in pathologic analyses. Eyes with vehicle B displayed more significant retinal changes including retinal hemorrhage, vascular narrowing, myelinated fiber edema, retinal necrosis and atrophy, and photoreceptor apoptosis. There was an increase in degree of the above damages as the volume of either vehicle increased. CONCLUSION: Both vehicle A and B caused volume-related toxicity in rabbit retina. The intensity of the toxic effects of different vehicles may differ. Reducing or decanting the vehicles should be considered before intravitreal use of TA injections.

Retinal toxicity of triamcinolone's vehicle (benzyl alcohol): an electrophysiologic and electron microscopic study.

PURPOSE: To assess retinal toxicity of the vehicle of triamcinolone, benzyl alcohol (BA), when injected into the vitreous cavity of rabbits. METHODS: This prospective comparative experimental study included 24 pigmented rabbits assigned into two groups: group 1 (experimental, n = 12) received intravitreal 0.1 ml of BA, and group 2 (control, n = 12) received intravitreal 0.1 ml of balanced salt solution (BSS); all injections were done in the right eyes. Clinical examinations [slit lamp biomicroscopy, indirect ophthalmoloscopy, and three intraocular pressure (IOP) measurements] were done on both eyes before injection, at 1 and 3 h post injection, together with electroretinograms (ERGs) at 3 days, 1, 2, 4, and 6 weeks following injections. Three rabbits from each group were euthanased at 1, 2, 4, or 6 weeks and eyes were sent for light and electron microscopic examination for quantitative morphometric measurements. RESULTS: The mean amplitudes of the a and b waves of the BA-injected eyes were 6.42 +/- 9.02 microv and 11.18 +/- 15.18 microv at 3 days, respectively, which were significantly reduced compared with the BSS-injected eyes (30.87 +/- 8.22 microv and 57.90 +/- 13.38 microv, respectively; P < 0.01 t-test) and the non-injected contralateral eyes (36.20 +/- 7.85 microv and 64.10 +/- 9.36 microv, respectively; P < 0.01 t-test). These ERG responses continued to be significantly reduced in the BA-injected eyes (P < 0.01 t-test) throughout the study period. The mean ganglion cell count was significantly reduced (P < 0.005 t-test) in the BA-injected eyes (8.42 +/- 2.4) compared with the BSS- and non-injected eyes (16.42 +/- 3.9 and 16.5 +/- 4.2, respectively). The mean thicknesses of the inner nuclear layer (INL) and outer nuclear layer (ONL) were significantly reduced (P < 0.005 t-test) in the BA-injected eyes (3.78 +/- 0.96 microm and 11.77 +/- 1.29 microm, respectively) compared with the BSS- (6.1 +/- 0.92 microm and 21.82 +/- 0.95 microm, respectively) and non-injected eyes (7.05 +/- 1.9 microm and 22.49 +/- 1.01 microm, respectively). Electron microscopy showed moderate to severe intracellular changes in the ganglion cell layer, INL, ONL, and photoreceptor layer at 6 weeks in BA-injected eyes, with no significant changes in BSS-injected eye. There was no significant rise in the IOP or clinical evidence of increased lens density during the study period in any of the eyes. CONCLUSIONS: Triamcinolone acetonide's vehicle, BA, produced severe ERG and structural damage to the retina when injected intravitreally.